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dna vaccine prime and replicating vaccinia vaccine boost induce robust humoral and cellular immune responses against MERS-CoV in mice

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Virologica Sinica 2024年第3期39卷 490-500页

作者： Xiuli Shen Shuhui Wang Yanling Hao Yuyu Fu Li Ren Dan Li Wenqi Tang Jing Li Ran Chen Meiling Zhu Shuo Wang Ying Liu Yiming Shao National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases National Center for AIDS/STD Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China Changping Laboratory Yard 28Science Park RoadChangping DistrictBeijing 102206China State Key Laboratory for Diagnosis and Treatment of Infectious Diseases National Clinical Research Center for Infectious DiseasesCollaborative Innovation Center for Diagnosis and Treatment of Infectious DiseasesThe First Affiliated HospitalSchool of MedicineZhejiang UniversityHangzhou 310003China

As of December 2022,2603 laboratory-identified Middle East respiratory syndrome coronavirus(MERS-CoV)infections and 935 associated deaths,with a mortality rate of 36%,had been reported to the World Health Organization(WHO).However,there are still no vaccines for MERS-CoV,which makes the prevention and control of MERS-CoV *** this study,we generated two dna vaccine candidates by integrating MERS-CoV Spike(S)gene into a replicating Vaccinia Tian Tan(VTT)*** to homologous immunization with either vaccine,mice immunized with dna vaccine prime and VTT vaccine boost exhibited much stronger and durable humoral and cellular immune *** immunized mice produced robust binding antibodies and broad neutralizing antibodies against the EMC2012,England1 and KNIH strains of ***-Boost immunization also induced strong MERS-S specific T cells responses,with high memory and poly-functional(CD107a-IFN-γ-TNF-α)effector CD8t T *** conclusion,the research demonstrated that dna-Prime/VTT-Boost strategy could elicit robust and balanced humoral and cellular immune responses against *** study not only provides a promising set of MERS-CoV vaccine candidates,but also proposes a heterologous sequential immunization strategy worthy of further development.

关键词： MERS-CoV VTT vaccine dna vaccine Humoral and cellular immune responses Prime/boost strategy

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dna vaccine of SARS-Cov S Gene Induces Antibody Response in Mice

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Acta Biochimica et Biophysica Sinica 2004年第1期36卷 37-41页

作者： Ping ZHAO, Jin-Shan KE, Zhao-Lin QIN, Hao REN, Lan-Juan ZHAO, Jian-Guo YU, Jun GAO, Shi-Ying ZHU, and Zhong-Tian QI (Department of Microbiology, Second Military Medical University, Shanghai 200433, China Department of Infectious Diseases, PLA Hospital No.88, Tai'an 271000, China ) Department of Microbiology Second Military Medical University Shanghai 200433 China.

The spike (S) protein, a main surface antigen of SARS-coronavirus (SARS-CoV), is one of themost important antigen candidates for vaccine design. In the present study, three fragments of the truncated S protein were expressed in E. coli, and analyzed with pooled sera of convalescence phase of SARS patients. The full length S gene dna vaccine was constructed and used to immunize BALB/c mice. The mouse serum IgG antibody against SARS-CoV was measured by ELISA with E. coli expressed truncated S protein or SARS-CoV lysate as diagnostic antigen. The results showed that all the three fragments of S protein expressed by E. coli was able to react with sera of SARS patients and the S gene dna candidate vaccine could induce the production of specific IgG antibody against SARS-CoV efficiently in mice with seroconversion ratio of 75% after 3 times of immunization. These findings lay some foundations for further understanding the immunology of SARS-CoV and developing SARS vaccines.

关键词： severe acute respiratory syndrome-coronavirus (SARS-CoV) spike protein dna vaccine antibody

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The Effect of Bovine IFN-α on the Immune Response in Guinea Pigs Vaccinated with dna vaccine of Foot-and-Mouth Disease Virus

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Acta Biochimica et Biophysica Sinica 2004年第10期36卷 701-706页

作者： Hui-Chen GUO, Zai-Xin LIU*, Shi-Qi SUN, Qing-Wen LENG1, Dong LI, Xiang-Tao LIU, and Qing-Ge XIE* Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China College of Animal Science and Technology, Shihezi University, Shihezi 832003, China 1 Key Laboratory of Animal Virology of Ministry of Agriculture Lanzhou Veterinary Research Institute Chinese Academy of Agricultural Sciences College of Animal Science and Technology Shihezi University

In this study, we constructed recombinant plasmid pcdna3.1/P12X3C3D including P1, 2A, 3C, 3D and part of 2B gene of FMDV and pcdna3.1/IFN containing the gene encoding bovine IFN-α. We inoculated the dna vaccine pcdna3.1/P12X3C3D with or without pcdna3.1/IFN to evaluate the effi- ciency of this dna vaccine and the immunogenicity of dna vaccine enhanced by the co-delivery with pcdna3.1/IFN. After two times of vaccination with dna vaccine, all of guinea pigs were challenged with 103 ID50 FMDV type O. Anti-FMDV antibody levels were detected by ELISA and T lymphocyte proliferation response was tested by MTT assay. The result shows that guinea pigs inoculated by pcdna3.1/P12X3C3D alone or with pcdna3.1/IFN generated specific antibodies and induced an FMDV-specific T lymphocyte proliferation response. FMDV challenge tests showed that one in four guinea pigs immunized by pcdna3.1/ P12X3C3D with pcdna3.1/IFN was protected from the FMDV serotype O infection. This result indicated that the efficiency of the dna vaccine was enhanced by co-delivery with pcdna3.1/IFN. However, the protection rate was considerably lower than that immunized with conventional FMD vaccine.

关键词： foot-and-mouth disease virus (FMDV) dna vaccine IFN-α gene vaccination

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Distribution and expression in vitro and in vivo of dna vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

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Chinese Journal of Oceanology and Limnology 2010年第1期28卷 67-74页

作者：郑风荣孙修勤刘洪展吴兴安钟楠王波周国栋 First Institute of Oceanography SOA Marine College Shandong University at Weihai Department of Microbiology Fourth Military Medical University Hainan Green Food Industrial company Ltd

Lymphocystis disease,caused by the lymphocystis disease virus (LCDV),is a significant worldwide problem in fish industry causing substantial economic *** this study,we aimed to develop the dna vaccine against LCDV,using dna vaccination *** evaluated plasmid pEGFP-N2-LCDV1.3 kb as a dna vaccine *** plasmid dna was transiently expressed after liposome transfection into the eukaryotic COS 7 cell *** distribution and expression of the dna vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR,RT-PCR and fluorescent *** from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle,the muscle opposite the injection site,the hind intestine,gill,spleen,head,kidney and liver,6 and 25 days after *** vaccine plasmids disappeared 100 d *** microscopy revealed green fluorescence in the injected muscle,the muscle opposite the injection site,the hind intestine,gill,spleen,head,kidney and liver of fish 48 h post-vaccination,green fluorescence did not appear in the control treated *** fluorescence became weak at 60 days ***-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6-50 days *** results demonstrate that the antigen encoded by the dna vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated *** antigen would therefore potentially initiate a specific immune *** plasmid dna was injected into Japanese flounder (Paralichthys olivaceus) intramuscularly and antibodies against LCDV were *** results indicate that the plasmid encoded dna vaccine could induce an immune response to LCDV and would therefore offer immune protection against *** studies are required for the development and application of this promising dna vaccine.

关键词： dna vaccine lymphocystis disease virus Paralichthys olivaceus

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Chitosan Microparticles Intended for Anti-caries dna vaccine Mucosal Delivery: Synthesis, Characterization and Transfection

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Journal of Wuhan University of Technology(Materials Science) 2005年第2期20卷 35-38页

作者：李宇红樊明文 Key Laboratory for Oral Biomedical Engineering Ministry of Education School of Stomatology Wuhan UniversityWuhan 430079 China Key Laboratory for Oral Biomedical Engineering Ministry of Education School of Stomatology Wuhan UniversityWuhan 430079 China Prof

In order to enhance the mucosal immunity of anti-caries dna vaccine, chitosan-dna microparticles for musocal vaccination were prepared by a coacervation method. The physicochemical structure of microparticles was investigated by a scanning electron microscope (SEM) and a cofocal laser scanning microscope (CLSM). For in-vitro studies, Hela cell was transfected by chitosan-dna *** expression of proteins was measured by the immunohistochemical methods, and the cytotocity of chitosan in Hela cell line was determined by the MTT assay. The experimental results show that the microparticles are about 2-6 μm in size and spherical in shape. The encapsulation efficiency is 99%, and the dna is almost captured in the micropraticles. Plasmid loaded into chitosan microparticles is distributed throughout these particles. The number of positive staining cells of chitosan-pGJA-P transfected cell is more than that of naked plasmid transfect cells, but less than that of Lipofect-dna complex group. Chitosan was found to be less cytotoxic compared with lipofectin (p<0.01).

关键词： chitosan microparticles anti-caries dna vaccine transfection

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Construction of sperm-specific lactate dehydrogenase dna vaccine and experimental study of its immuno-contraceptive effect on mice

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Science China(Life Sciences) 2008年第4期51卷 308-316页

作者： CHEN Yong ZHANG Duo XIN Na XIONG YongZhong CHEN Ping LI Bo TU XiangDong LAN FengHua Research Laboratory of Medical Genetics PLA Institute of Technology for Birth ControlFetal Health and Child GrowthFuzhou General Hospital of Nanjing Military RegionFuzhou 350025China

Lactate dehydrogenase C4 (LDHC4) is a key enzyme for sperm metabolism. It is distributed specifically in testis and is highly immunogenic. In this study, two dna vaccines pVAX1-hLDHC and pVAX1-mLDHC were constructed by inserting coding sequences of human and mice LDHC4 into the eukaryotic ex-pression vector pVAX1. The production of LDHC4 specific antibodies was induced in the sera of vac-cinated mice and the reproductive tract secretions of vaccinated female mice through immunization by mucosal surface instillation. Furthermore, the antibody titer increased with the times of immunization. In the mating experiment, the number of newborns of the vaccinated mice reduced significantly and some immunized female mice even lost the ability to bear any offsprings, suggesting that the difference between the immunized and control mice was statistically significant. Sperm agglutination analysis indicated that both the antisera from immunized mice and the reproductive tract secretions of vacci-nated female mice could agglutinate normal sperms. Results of immunohistochemistry showed that the antibodies present in the sera of immunized mice and the reproductive tract secretions of vaccinated female mice could specifically react with LDHC4 antigen, which mainly locates in the cytoplasm, acrosome membrane externa and acrosome capsule of the sperm. Taken together, our results indicated that the constructed contraceptive dna vaccines did yield immunocontraceptive effects on mice and this would enable clinical trials in near future.

关键词： sperm-specific lactate dehydrogenase dna vaccine contraceptive vaccine mucosal immunization

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HPV16E7-HSP70 Hybrid dna vaccine Induces E7-Specific Cytotoxic T Cells and Antitumor Immunity

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Wuhan University Journal of Natural Sciences 2006年第3期11卷 749-755页

作者： ZHU Liqin LI Hui XIONG Jinhu WANG Tongxiang OU Xuan WEI Yun WU Xinxing Institute of Virology Medical School Wuhan University Wuhan 430071 Hubei China

Using human papillomavirus type 16 （HPV16） E7 as an antigen and Heat Shock Protein 70 as adjuvant, we constructed a dna vaccine by linking HSP70 gene to E7^C91G; gene. Mice, after being immunized with E7^C91G;-HSP70, E7^C91G/HSP70, E7^C91G, and wild E7 dna vaccines respectively, produced E7 specific CD8^＋ T-cell precursor frequencies of 280.33±2.52, 144.34±4.04, 164.34±5.13 and 82.33±3.51 respectively within every 1 × 10^5 mouse splenocytes. This proves that E7^C91G-HSP70 fusion vaccine can significantly enhance the E7 specific cellular immunity within the mice body（p〈0. 01）. After being immunized with E7^C91G-HSP70 fusion vaccine, tumor-bearing mice of the group being treated have significantly longer latency and survival periods, comparing with other three categories of E7 vaccines. Experiment shows that this vaccine has a significant effect on enhancing E7 positive tumor-treatment within mice body. After being immunized with E7^C91G-HSP70 vaccine, there were no pathological changes found in livers, kidneys and spleens of the mice, which proves that the vaccine is quite safe. After all, E7^C91G-HSP70 fusion vaccine has a much stronger tumor- treatment effect than thai of wild type E7 dna vaccine.

关键词： human papillomavirus type 16 (HPV16) E7 gene dna vaccine cellular immunity

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Interleukin-12 as a Genetic Adjuvant Enhances Hepatitis C Virus NS3 dna vaccine Immunogenicity

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Virologica Sinica 2013年第3期28卷 167-173页

作者： Malihe Naderi Atefeh Saeedi Abdolvahab Moradi Mishar Kleshadi Mohammad Reza Zolfaghari Ali Gorji Amir Ghaemi Department of Microbiology Qom branch Islamic Azad University Department of Microbiology Lahijan Branch Islamic Azad University Golestan Research Center of Gastroenterology and Hepatology-GRCGH Department of Microbiology Golestan University of Medical Sciences Shefa Neuroscience Research Centre Institut für Physiologie I Westflische Wilhelms-Universitt Münster Klinik und Poliklinik für Neurochirurgie Westf lische Wilhelms-Universitt Münster

Hepatitis C virus (HCV) chronic infection is a worldwide health problem, and numerous efforts have been invested to develop novel vaccines. An efficient vaccine requires broad immune response induction against viral proteins. To achieve this goal, we constructed a dna vaccine expressing nonstructural 3 (NS3) gene (pcdna3.1-HCV-NS3) and assessed the immune response in C57BL/6 mice. In this study, the NS3 gene was amplified with a nested-reverse transcriptase-polymerase chain reaction (RT-PCR) method using sera of HCV-infected patients with genotype 1a. The resulting NS3 gene was subcloned into a pcdna3.1 eukaryotic expression vector, and gene expression was detected by western blot. The resultant dna vaccine was co-administered with interleukin-12 (IL-12) as an adjuvant to female C57BL/6 mice. After the final immunizations, lymphocyte proliferation, cytotoxicity, and cytokine levels were assessed to measure immune responses. Our data suggest that co-administration of HCV NS3 dna vaccine with IL-12 induces production of significant levels of both IL-4 and interferon (IFN)-γ (p<0.05). Cytotoxicity and lymphocyte proliferation responses of vaccinated mice were significantly increased compared to control (p<0.05). Collectively, our results demonstrated that co-administration of HCV NS3 and IL-12 displayed strong immunogenicity in a murine model.

关键词： Hepatitis C virus (HCV) NS3 Interleukin-12 dna vaccine

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In Vivo Kinetics and Biodistribution of a Hantaan Virus dna vaccine after Intramuscular Injection in Mice

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Virologica Sinica 2010年第3期25卷 177-182页

作者： Si WANG Qing NIE Lan-yan ZHENG Jun HU En-jie LUO Department of Medical microbiology and parasitology Preclinical Medical College China Medical University Shenyang 110001 China

To study the kinetics in vivo of a Hantaan virus dna vaccine, we constructed a fusion dna vaccine, pEGFP/S, by cloning the S segment of Hantavirus into the vector, pEGFP-C1, which encodes Green fluorescent protein EGFP. In this report, we provide evidence that pEGFP/S was distributed and persistently expressed for more than 60 days in several organs after inoculation. Our findings suggest that the persistent immune responses induced by a Hantaan virus dna vaccine are likely due to the plasmid pEGFP/S deposited in vivo, which acts as a booster immunization.

关键词： Hantaan virus dna vaccine Intramuscular injection Immunologic memory Nucleocapsid protein

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Enhancement Effect of CpG dna on the Somatostatin dna vaccine in Mice

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Agricultural Sciences in China 2007年第7期6卷 882-889页

作者： XUE Chun-lin MAO Da-gan YANG Li-guo CHENG Bao College of Animal Science and Technology Huazhong Agricultural University Wuhan 430070 P.R.China Research Institute of Animal Breeding ＆ Reproduction Nanjing Agricultural University Nanjing 210095 P.R.China Yancheng Institute of Animal Husbandry ＆ Veterinary Yancheng 224002 P.R. China

To study the immune effect of CpGdna on somatostatin （SS） dna vaccine, the 20-day-old experimental mice were immunized with 20 lag SS eukaryotic expression plasmid pES/2SS with different adjuvants in equal dose, such as the synthetic CpG-ODN, the pE-CpG plasmid, E. coli dna and the crude liposome. A booster was given two weeks later. The results showed that the body weight gain of female mice in the SS immunized group was higher than that of the control （P 〈0.05）. The levels of antibodies against SS, IgG2a/IgG1, spleen lymphocyte proliferation activity and the concentrations of GH and IGF-Ⅰ in the dna vaccine groups combined with CpGdna were significantly increased compared to that of the group immunized with dna vaccine alone. All these suggested the recombinant SS expression plasmid can stimulate animals to produce antibodies against SS, and CpGdna adjuvant can enhance the immune effect of dna vaccine against SS and influence the concentration of GH and IGF-Ⅰ .

关键词： immnostimulatory sequence CpGdna somatostatin dna vaccine

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