Although a prophylactic vaccine is available,hepatitis B virus(HBV)remains a major cause of liver-related morbidity and mortality.Current treatment options are improving clinical outcomes in chronic hepatitis B;howeve...Although a prophylactic vaccine is available,hepatitis B virus(HBV)remains a major cause of liver-related morbidity and mortality.Current treatment options are improving clinical outcomes in chronic hepatitis B;however,true functional cure is currently the exception rather than the rule.Nucleic acid vaccines are among the emerging immunotherapies that aim to restore weakened immune function in chronically infected hosts.DNA vaccines in particular have shown promising results in vivo by reducing viral replication,breaking immune tolerance in a sustained manner,or even decimating the intranuclear covalently closed circular DNA reservoir,the hallmark of HBV treatment.Although DNA vaccines encoding surface antigens administered by conventional injection elicit HBVspecific T cell responses in humans,initial clinical trials failed to demonstrate additional therapeutic benefit when administered with nucleos(t)ide analogs.In an attempt to improve vaccine immunogenicity,several techniques have been used,including codon/promoter optimization,coadministration of cytokine adjuvants,plasmids engineered to express multiple HBV epitopes,or combinations with other immunomodulators.DNA vaccine delivery by electroporation is among the most efficient strategies to enhance the production of plasmid-derived antigens to stimulate a potent cellular and humoral anti-HBV response.Preliminary results suggest that DNA vaccination via electroporation efficiently invigorates both arms of adaptive immunity and suppresses serum HBV DNA.In contrast,the study of mRNA-based vaccines is limited to a few in vitro experiments in this area.Further studies are needed to clarify the prospects of nucleic acid vaccines for HBV cure.展开更多
BACKGROUND: The deposition of α -synuclein ( α -syn) aggregates is a neuropathological feature of Parkinson's disease. It remains impossible to involve α-syn aggregation in the treatment of Parkinson's dise...BACKGROUND: The deposition of α -synuclein ( α -syn) aggregates is a neuropathological feature of Parkinson's disease. It remains impossible to involve α-syn aggregation in the treatment of Parkinson's disease. A nucleic acid vaccine will provide a new pathway to immunotherapy for Parkinson's disease. OBJECTIVE: To construct a recombinant eukaryotic expression vector pVAX1 coding human α -syn and to observe its expression level in COS-7 cells. DESIGN AND SETTING: The present bioengineering and molecular biology experiment was performed at Department of Neurology, First Affiliated Hospital of Chongqing Medical University & Chongqing Key Laboratory of Neurology. MATERIALS: The eukaryotic expression plasmid pVAXI, human embryonic brain tissue, healthy human blood cells, and COS-7 cells were purchased from Promega Company, USA. METHODS: The full-length CDS sequence of the human a -syn gene was amplified by RT-PCR, which contained restriction sites for the enzymes Kpn Ⅰ, Xba Ⅰ and Kozak consensus sequence. Then the PCR products and eukaryotic expression vector pVAX1 were digested with Kpn Ⅰ and Xba Ⅰ simultaneously, and were extracted and ligated by T4 ligase. The recombinant constructs pVAX1-h α -S1-140 were transformed into competent E. coli TOP 1 0 cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis, and DNA sequencing. The constructs were then tested for protein expression in COS-7 cells by RT-PCR and Western blotting. MAIN OUTCOME MEASURES: Identification of an eukaryotic expression vector containing the human α -syn gene, pVAX1-h α-S1-140, and detection of the expression in mammalian cell COS-7. RESULTS: The pVAX1 vector was successfully cloned with human α -syn in the correct orientation and in-frame. The DNA vaccine constructs pVAX 1-h α-S1-140 with the human α-syn gene were shown to be expressed in COS-7 cells. Human α-syn was successfully expressed in the mammalian cell line and was detected by RT-PCR and western blotting. CONCLUSION: Nucleic acid vaccine pVAX1-h α S1-140 was successfully constructed and expressed in COS-7 cells.展开更多
In order to develop swine hepatitis E (HE) genetically engineering vaccines, specific primers of genes LB1, LB2, LB3 of swine hepatitis E virus were designed and used for amplification, DNA amplieons generated by PC...In order to develop swine hepatitis E (HE) genetically engineering vaccines, specific primers of genes LB1, LB2, LB3 of swine hepatitis E virus were designed and used for amplification, DNA amplieons generated by PCR assays were directly cloned into T-A plasmid and expressed using pEASY-M1 expression vector. Three recombinant eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3 were constructed. The eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2, and pEASY-LB3 were transfected into 293T cells, and three target genes were detected by real-time fluorescent quantitative RT-PCR. The results confirmed that three eukaryotic expression plasmids were transfected into 293Teells and target protein was expressed. Analysis by SDS-PAGE electrophoresis and Western-blot indicated that three target proteins were expressed in 293T cells transfected with eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3. Antigenicity studies indicated good HEV responses. Therefore, three recombinant DNAs of HEV ORF2 nucleic acid vaccine candidates were ob- tained, which might lay the foundation for further studies in the future.展开更多
Nucleic acid vaccine or DNA vaccine is a hopeful vac- cine to prevent and treat viral hepatitis. Problems exist in different DNA vaccines for HBV or HCV. Optimal animal model should be established study vaccine agains...Nucleic acid vaccine or DNA vaccine is a hopeful vac- cine to prevent and treat viral hepatitis. Problems exist in different DNA vaccines for HBV or HCV. Optimal animal model should be established study vaccine against hepatitis. Apart from the strategy to enhance the efficiency of DNA vaccine, combined use of cytokines or chemokines, different routes of inocu- lation, design of optimal vector, ISS insertion in the plasmid vectors, etc to enhance the efficiency of DNA vaccine are reviewed.展开更多
The coronavirus disease 2019(COVID-19)mortality rate in 55 African countries is almost 4.5 times lower than in the coronavirus disease 2019(COVID-19)despite Africa having over 4.2 times more people.This mortality para...The coronavirus disease 2019(COVID-19)mortality rate in 55 African countries is almost 4.5 times lower than in the coronavirus disease 2019(COVID-19)despite Africa having over 4.2 times more people.This mortality paradox is also evident when comparing Nigeria,a heavily populated,poorly vaccinated and weakly mandated country to Israel,a small,highly vaccinated and strictly mandated country.Nigeria has almost 4 times lower COVID mortality than Israel.In this Field of Vision perspective,I explain how this paradox has evolved drawing upon my academic,clinical and social experience.Since April 2020,I’ve developed and been using the Egyptian immune-modulatory Kelleni’s protocol to manage COVID-19 patients including pediatric,geriatric,pregnant,immune-compromised and other individuals suffering from multiple comorbidities.It’s unfortunate that severe acute respiratory syndrome coronavirus 2 is still evolving accompanied by more deaths.However in Africa,we’ve been able to live without anxiety or mandates throughout the pandemic because we trust science and adopted early treatment using safe,and effective repurposed drugs that have saved the majority of COVID-19 patients.This article represents an African and Egyptian tale of honor.展开更多
Objective: To explore the antitumor effects of hemaagglutinin-neuraminase gene (HN gene) from Newcastle disease virus. Methods: Plasmid vaccine of pIRHN was constructed and transfected into HeLa cells. The expression...Objective: To explore the antitumor effects of hemaagglutinin-neuraminase gene (HN gene) from Newcastle disease virus. Methods: Plasmid vaccine of pIRHN was constructed and transfected into HeLa cells. The expression of HN was analyzed by Western blot analysis, and the mode of cell death was detected by fluorescence microscope, gel electrophoresis and TUNEL assay and the expression of p53 and bcl-2 was also analyzed in transfected Hela cells. The effect of pIRHN on sialic acid contents in the Hela cell was examined. Results: pIRHN nucleic acid vaccines could be expressed in eukaryotic cell. pIRHN could induce apoptosis after HeLa cells were transfected. The effect of antitumor responses of pIRHN was correlated with the contents of sialic acid in tumor cells, and there was no prominent evidence for the relatedness of the antitumor effect with the expression of p53 and bcl-2. Conclusion: pIRHN may become a new antitumor biological agent.展开更多
Hepatitis C virus(HCV)is responsible for no less than 71 million people chronically infected and is one of the most frequent indications for liver transplanta-tion worldwide.Despite direct-acting antiviral therapies f...Hepatitis C virus(HCV)is responsible for no less than 71 million people chronically infected and is one of the most frequent indications for liver transplanta-tion worldwide.Despite direct-acting antiviral therapies fuel optimism in controlling HCV infections,there are several obstacles regarding treatment accessibility and reinfection continues to remain a possibility.Indeed,the majority of new HCV infections in developed countries occur in people who inject drugs and are more plausible to get reinfected.To achieve global epidemic control of this virus the development of an effective prophylactic or therapeutic vaccine becomes a must.The coronavirus disease 19(COVID-19)pandemic led to auspicious vaccine development against severe acute respiratory syndrome coronavirus-2(SARSCoV-2)virus,which has renewed interest on fighting HCV epidemic with vaccination.The aim of this review is to highlight the current situation of HCV vaccine candidates designed to prevent and/or to reduce HCV infectious cases and their complications.We will emphasize on some of the crossroads encountered during vaccine development against this insidious virus,together with some key aspects of HCV immunology which have,so far,ham-pered the progress in this area.The main focus will be on nucleic acid-based as well as recombinant viral vector-based vaccine candidates as the most novel vaccine approaches,some of which have been recently and successfully employed for SARS-CoV-2 vaccines.Finally,some ideas will be presented on which methods to explore for the design of live-attenuated vaccines against HCV.展开更多
Malignant tumors are still a worldwide threat to human health.Tumor treatment strategies are constantly evolving,and the advent of tumor immunotherapy has brought up hope to many types of tumors,especially for those t...Malignant tumors are still a worldwide threat to human health.Tumor treatment strategies are constantly evolving,and the advent of tumor immunotherapy has brought up hope to many types of tumors,especially for those that are refractory to conventional therapies including surgery,radiotherapy,and chemotherapy.Tumor vaccines can initiate or amplify an anti-tumor immune response in tumor patients through active immunization,and therefore occupy an important position in tumor immunotherapy.The main types of tumor vaccines include tumor cell vaccines,dendritic cell vaccines,polypeptide vaccines and nucleic acid vaccines.Due to factors such as poor antigen selection and suppressive tumor microenvironment,earliest tumor vaccines on clinical trials failed to achieve satisfactory clinical effects.However,with the development of second-generation genome sequencing technologies and bioinformatics tools,it is possible to predict neoantigens generated by tumor-specific mutations and therefore prepare personalized vaccines.This article summarizes the global efforts in developing tumor vaccines and highlights several representative tumor vaccines in each category.展开更多
Objective: To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG ...Objective: To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG and boosting with plasmid DNA pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD 4 + T cells from splenic lymphocytes increased significantly and the proliferation response of splenocytes to ConA and LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity responses in mice.展开更多
The CP15/60 gene encoding the CP15/60 surface protein of sporozoites in Cryptosporidiumparvum was obtained by PCR so as to research the nucleic vaccine against C.parvum. Theeukaryotic expressing vector pcDNA3-15/60 wa...The CP15/60 gene encoding the CP15/60 surface protein of sporozoites in Cryptosporidiumparvum was obtained by PCR so as to research the nucleic vaccine against C.parvum. Theeukaryotic expressing vector pcDNA3-15/60 was constructed by inserting CP15/60 gene intopcDNA3 (+) in XhoⅠand EcoRⅠ. A vaccination protocol was the adult pregnant goatsinoculated intranasally with the pcDNA3-15/60 plasmid and their offspring were infectedwith C.parvum oocysts. The results showed that the pcDNA3-15/60 plasmid can induce theimmune response of goats and the vaccinated goats can transfer the immunity to offspringconferring protection against C.parvum infection. These suggested that the recombinantplasmid could be a DNA vaccine candidate.展开更多
文摘Although a prophylactic vaccine is available,hepatitis B virus(HBV)remains a major cause of liver-related morbidity and mortality.Current treatment options are improving clinical outcomes in chronic hepatitis B;however,true functional cure is currently the exception rather than the rule.Nucleic acid vaccines are among the emerging immunotherapies that aim to restore weakened immune function in chronically infected hosts.DNA vaccines in particular have shown promising results in vivo by reducing viral replication,breaking immune tolerance in a sustained manner,or even decimating the intranuclear covalently closed circular DNA reservoir,the hallmark of HBV treatment.Although DNA vaccines encoding surface antigens administered by conventional injection elicit HBVspecific T cell responses in humans,initial clinical trials failed to demonstrate additional therapeutic benefit when administered with nucleos(t)ide analogs.In an attempt to improve vaccine immunogenicity,several techniques have been used,including codon/promoter optimization,coadministration of cytokine adjuvants,plasmids engineered to express multiple HBV epitopes,or combinations with other immunomodulators.DNA vaccine delivery by electroporation is among the most efficient strategies to enhance the production of plasmid-derived antigens to stimulate a potent cellular and humoral anti-HBV response.Preliminary results suggest that DNA vaccination via electroporation efficiently invigorates both arms of adaptive immunity and suppresses serum HBV DNA.In contrast,the study of mRNA-based vaccines is limited to a few in vitro experiments in this area.Further studies are needed to clarify the prospects of nucleic acid vaccines for HBV cure.
文摘BACKGROUND: The deposition of α -synuclein ( α -syn) aggregates is a neuropathological feature of Parkinson's disease. It remains impossible to involve α-syn aggregation in the treatment of Parkinson's disease. A nucleic acid vaccine will provide a new pathway to immunotherapy for Parkinson's disease. OBJECTIVE: To construct a recombinant eukaryotic expression vector pVAX1 coding human α -syn and to observe its expression level in COS-7 cells. DESIGN AND SETTING: The present bioengineering and molecular biology experiment was performed at Department of Neurology, First Affiliated Hospital of Chongqing Medical University & Chongqing Key Laboratory of Neurology. MATERIALS: The eukaryotic expression plasmid pVAXI, human embryonic brain tissue, healthy human blood cells, and COS-7 cells were purchased from Promega Company, USA. METHODS: The full-length CDS sequence of the human a -syn gene was amplified by RT-PCR, which contained restriction sites for the enzymes Kpn Ⅰ, Xba Ⅰ and Kozak consensus sequence. Then the PCR products and eukaryotic expression vector pVAX1 were digested with Kpn Ⅰ and Xba Ⅰ simultaneously, and were extracted and ligated by T4 ligase. The recombinant constructs pVAX1-h α -S1-140 were transformed into competent E. coli TOP 1 0 cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis, and DNA sequencing. The constructs were then tested for protein expression in COS-7 cells by RT-PCR and Western blotting. MAIN OUTCOME MEASURES: Identification of an eukaryotic expression vector containing the human α -syn gene, pVAX1-h α-S1-140, and detection of the expression in mammalian cell COS-7. RESULTS: The pVAX1 vector was successfully cloned with human α -syn in the correct orientation and in-frame. The DNA vaccine constructs pVAX 1-h α-S1-140 with the human α-syn gene were shown to be expressed in COS-7 cells. Human α-syn was successfully expressed in the mammalian cell line and was detected by RT-PCR and western blotting. CONCLUSION: Nucleic acid vaccine pVAX1-h α S1-140 was successfully constructed and expressed in COS-7 cells.
基金Supported by the Basal Research Fund of Guangxi(10-111-1)+2 种基金the Guangxi Science and Technology Project(10100014-4)the Scientific Research Project of Guangxi Bureau of Livestock,Fisheries and Veterinary Services(12049031)the Systemic Research Project of Guangxi Key Laboratory of Animal Vaccines and New Technology(12-071-28-A-5)
文摘In order to develop swine hepatitis E (HE) genetically engineering vaccines, specific primers of genes LB1, LB2, LB3 of swine hepatitis E virus were designed and used for amplification, DNA amplieons generated by PCR assays were directly cloned into T-A plasmid and expressed using pEASY-M1 expression vector. Three recombinant eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3 were constructed. The eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2, and pEASY-LB3 were transfected into 293T cells, and three target genes were detected by real-time fluorescent quantitative RT-PCR. The results confirmed that three eukaryotic expression plasmids were transfected into 293Teells and target protein was expressed. Analysis by SDS-PAGE electrophoresis and Western-blot indicated that three target proteins were expressed in 293T cells transfected with eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3. Antigenicity studies indicated good HEV responses. Therefore, three recombinant DNAs of HEV ORF2 nucleic acid vaccine candidates were ob- tained, which might lay the foundation for further studies in the future.
文摘Nucleic acid vaccine or DNA vaccine is a hopeful vac- cine to prevent and treat viral hepatitis. Problems exist in different DNA vaccines for HBV or HCV. Optimal animal model should be established study vaccine against hepatitis. Apart from the strategy to enhance the efficiency of DNA vaccine, combined use of cytokines or chemokines, different routes of inocu- lation, design of optimal vector, ISS insertion in the plasmid vectors, etc to enhance the efficiency of DNA vaccine are reviewed.
文摘The coronavirus disease 2019(COVID-19)mortality rate in 55 African countries is almost 4.5 times lower than in the coronavirus disease 2019(COVID-19)despite Africa having over 4.2 times more people.This mortality paradox is also evident when comparing Nigeria,a heavily populated,poorly vaccinated and weakly mandated country to Israel,a small,highly vaccinated and strictly mandated country.Nigeria has almost 4 times lower COVID mortality than Israel.In this Field of Vision perspective,I explain how this paradox has evolved drawing upon my academic,clinical and social experience.Since April 2020,I’ve developed and been using the Egyptian immune-modulatory Kelleni’s protocol to manage COVID-19 patients including pediatric,geriatric,pregnant,immune-compromised and other individuals suffering from multiple comorbidities.It’s unfortunate that severe acute respiratory syndrome coronavirus 2 is still evolving accompanied by more deaths.However in Africa,we’ve been able to live without anxiety or mandates throughout the pandemic because we trust science and adopted early treatment using safe,and effective repurposed drugs that have saved the majority of COVID-19 patients.This article represents an African and Egyptian tale of honor.
基金This work was supported by the National 973 Basic Research Development Program of China (No. G199011902).
文摘Objective: To explore the antitumor effects of hemaagglutinin-neuraminase gene (HN gene) from Newcastle disease virus. Methods: Plasmid vaccine of pIRHN was constructed and transfected into HeLa cells. The expression of HN was analyzed by Western blot analysis, and the mode of cell death was detected by fluorescence microscope, gel electrophoresis and TUNEL assay and the expression of p53 and bcl-2 was also analyzed in transfected Hela cells. The effect of pIRHN on sialic acid contents in the Hela cell was examined. Results: pIRHN nucleic acid vaccines could be expressed in eukaryotic cell. pIRHN could induce apoptosis after HeLa cells were transfected. The effect of antitumor responses of pIRHN was correlated with the contents of sialic acid in tumor cells, and there was no prominent evidence for the relatedness of the antitumor effect with the expression of p53 and bcl-2. Conclusion: pIRHN may become a new antitumor biological agent.
基金Supported by Programa de Desarrollo de las Ciencias Basicas(PEDECIBA)Comision Academica de Posgrados,Universidad de la Republica Uruguay(UdelaR)Comision Sectorial de Investigacion Cientifica(CSIC,I+D Project ID288).
文摘Hepatitis C virus(HCV)is responsible for no less than 71 million people chronically infected and is one of the most frequent indications for liver transplanta-tion worldwide.Despite direct-acting antiviral therapies fuel optimism in controlling HCV infections,there are several obstacles regarding treatment accessibility and reinfection continues to remain a possibility.Indeed,the majority of new HCV infections in developed countries occur in people who inject drugs and are more plausible to get reinfected.To achieve global epidemic control of this virus the development of an effective prophylactic or therapeutic vaccine becomes a must.The coronavirus disease 19(COVID-19)pandemic led to auspicious vaccine development against severe acute respiratory syndrome coronavirus-2(SARSCoV-2)virus,which has renewed interest on fighting HCV epidemic with vaccination.The aim of this review is to highlight the current situation of HCV vaccine candidates designed to prevent and/or to reduce HCV infectious cases and their complications.We will emphasize on some of the crossroads encountered during vaccine development against this insidious virus,together with some key aspects of HCV immunology which have,so far,ham-pered the progress in this area.The main focus will be on nucleic acid-based as well as recombinant viral vector-based vaccine candidates as the most novel vaccine approaches,some of which have been recently and successfully employed for SARS-CoV-2 vaccines.Finally,some ideas will be presented on which methods to explore for the design of live-attenuated vaccines against HCV.
文摘Malignant tumors are still a worldwide threat to human health.Tumor treatment strategies are constantly evolving,and the advent of tumor immunotherapy has brought up hope to many types of tumors,especially for those that are refractory to conventional therapies including surgery,radiotherapy,and chemotherapy.Tumor vaccines can initiate or amplify an anti-tumor immune response in tumor patients through active immunization,and therefore occupy an important position in tumor immunotherapy.The main types of tumor vaccines include tumor cell vaccines,dendritic cell vaccines,polypeptide vaccines and nucleic acid vaccines.Due to factors such as poor antigen selection and suppressive tumor microenvironment,earliest tumor vaccines on clinical trials failed to achieve satisfactory clinical effects.However,with the development of second-generation genome sequencing technologies and bioinformatics tools,it is possible to predict neoantigens generated by tumor-specific mutations and therefore prepare personalized vaccines.This article summarizes the global efforts in developing tumor vaccines and highlights several representative tumor vaccines in each category.
基金This work was supported by the National Scientific Research Foundation for Excellent Young Scientist of China (No. 398251197).
文摘Objective: To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG and boosting with plasmid DNA pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD 4 + T cells from splenic lymphocytes increased significantly and the proliferation response of splenocytes to ConA and LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity responses in mice.
基金supported by Natural Science Foundation of Jilin Province for Outstanding Young scientists Award,China(2002).
文摘The CP15/60 gene encoding the CP15/60 surface protein of sporozoites in Cryptosporidiumparvum was obtained by PCR so as to research the nucleic vaccine against C.parvum. Theeukaryotic expressing vector pcDNA3-15/60 was constructed by inserting CP15/60 gene intopcDNA3 (+) in XhoⅠand EcoRⅠ. A vaccination protocol was the adult pregnant goatsinoculated intranasally with the pcDNA3-15/60 plasmid and their offspring were infectedwith C.parvum oocysts. The results showed that the pcDNA3-15/60 plasmid can induce theimmune response of goats and the vaccinated goats can transfer the immunity to offspringconferring protection against C.parvum infection. These suggested that the recombinantplasmid could be a DNA vaccine candidate.