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172. Characterization of Immune Response in Mice Induced by Recombinant Vaccinia Virus (Tiantan) Based Multivalent H5N1 Avian Influenza Vaccines
INFECTIOUS DISEASES AND VACCINES: VACCINE RELATED GENE TRANSFER RESEARCH measles, mumps and rubella and has an excellent safety record after millions of doses administered in children. On the other hand, MV is an attractive vector platform for expression of foreign antigens and vaccine development. Here we present characterization and immune response to MV strains encoding HP-NAP antigen. Results: HP-NAP from two different H. pylori strains was cloned into the MV Edmoston vector platform (MV-NAP-26695 and MVNAP-43504) in position upstream of N gene. To facilitate production and extracellular secretion of the protein, the NAP gene was inserted in the human lambda light immunoglobulin chain replacing a large part of variable domain. We generated MV strains expressing two different secretory NAP forms: MV-lambda-NAP (encoding the full length constant lambda domain) and MV-s-NAP (encoding only the N-terminus of immunoglobulin molecule with the leader peptide). Both vectors expressed high levels of the secretory NAP antigen (> 4µg/106 infected cells). Growth kinetics data showed that the NAP insert did not interfere and did not inhibit viral replication. Secretion of biologically active NAP by MV-s-NAP infected cells triggered signicant Th1 cytokine and IL-8 production in THP-1 monocytic cells. A singe i.p. injection of NAP-expressing strains induced a robust humoral and cellular response against MV in Ifnarko-CD46Ge transgenic mice. There was no signicant difference in MV-specic antibody response between mice immunized with NAP-expressing MV strains and those injected with control MV-GFP (MV expressing green uorescent protein). The anti-measles immunity was longlasting and after 9 months the serum neutralizing titer was still above level considered protective for humans (>1:120 PNT50). The animals immunized with MV-lambda-NAP and MV-s-NAP developed stronger anti-NAP response compared to MV-NAP injected groups. The NAP-specic antibody titer reached the peak (>1:10,000 in ELISA) 2-4 weeks post vaccination. In addition, puried HP-NAP antigen stimulated IFN-γ expression in lymphocytes from immune animals indicating that NAP-encoding MV vectors induced also NAP-specic cell-mediated immunity. Conclusions: Our data demonstrated that MV is an excellent platform for expression of bacterial antigens and development of vaccines against H. pylori. Expression of HP-NAP does not affect virus replication and induction of protective anti-measles immunity. In contrast to puried H. pylori antigen formulated vaccines, MVs encoding secretory NAP induced strong humoral and cellular immune response. * This work was supported by Atwater grant, P50CA116201 and Paul Leibson Memorial Fund.
171. Generation of Chimeric GB Virus B Clones Encoding the Hepatitis C Virus E1E2p7 and coreE1E2p7 toward the Infected Marmoset Models
Tingting Li,1 Lifang Shuai,1 Zixuan Chen,1 Anqi Wang,1 Wenjing Wang,1 Chengyao Li.1 1 School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China.
The development of novel therapy and vaccination for hepatitis C virus (HCV) has been hampered by a lack of a small primate model. GB virus B (GBV-B) is closely related to HCV that causes acute or chronic hepatitis in marmosets, and thus, is an attractive surrogate model for HCV. Even more attractive, for mostly mimicking HCV infection in marmoset models, is the idea of constructing chimeric viruses by replacing GBV-B elements with its HCV counterpart of interest. Two infectious GBV-B chimeric genomes, HCV E1E2p7/ GBV-B and HCV coreE1E2p7/GBV-B, containing the coding region of E1 E2 p7 proteins and core E1 E2 p7 proteins were constructed, respectively. Overlapping PCR and restriction-enzyme digestion methods were used to generate the chimeric plasmids and then RNA transcripts were synthesized in vitro. The chimera may prove useful for establishing HCV infected marmoset models and determining HCV epitopes and immune responses in vivo. S66
172. Characterization of Immune Response in Mice Induced by Recombinant Vaccinia Virus (Tiantan) Based Multivalent H5N1 Avian Inuenza Vaccines
Wen Wang, Yao Deng, Wenjie Tan, Hong Chen, Li Ruan, Yuelong Shu. National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China. To develop an effective and broad immune protective H5N1 vaccine, We rst developed two recombinant vaccinia(Tiantan strain) virus(rTTV) based H5N1 vaccines, which consisted of bicistron expressing the hemagglutinin(HA) and matrix protein 2(M2),or bicistron expressing the neuraminidase(NA) and matrix protein 1(M1). The expression of H5N1 protein in rTTVs was conrmed. We immunized the Balb/C mice twice with two kind of dose (104 pfu, 107 pfu) using different combination. Subsequently, we assessed the humoral and cellular immune response in vaccinated mice. Our data showed that rTTV-based H5N1 vaccine induced rapidly robust HAand NA-specic antibody level and IFNγ secreting form cell(SFC) with either single dose of 107 pfu or twice dose of 104 pfu or 107 pfu. We also detected signicant neutralizing antibody and matrix-specic immune response. In addition, we found that immunization with two kind of rTTV-based H5N1 vaccines induced much high level of matrix 2-specic antibody than that with single of rTTV-based H5N1 vaccine. In conclusion, rTTV-based H5N1 vaccines elicit board array of immunity and our study offers a promising alternative H5N1 prevaccine candidates with favorable potential to prevent various H5N1 pandemic.
173. The Effects of the Calreticulin on the Immune Response Induced by DNA Vaccine Containing the Classical Swine Fever Virus E2 Gene in Mouse Model
Ming Kun Hsieh, Chia Chin You, Chienjin Huang. Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan. The structural glycoprotein E2 of the Classical swine fever virus (CSFV) can stimulate the production of neutralizing antibody which can protect pigs against CSF. Previous reports showed that DNA vaccine containing the E2 gene of CSFV was able to provide protective immunity. Calreticulin (CRT) has demonstrated the ability to enhance MHC class I presentation and elicit antigen-specic CD8+ T cell immune responses. The purpose of this study was to determine whether the mouse CRT gene can enhance the immune response of DNA vaccine by fusing with the E2 gene of CSFV in mouse model. Protein expression of plasmids containing CSFV E2 gene (pcDNA4/E2), mouse CRT gene (pcDNA4/CRT) or both (pcDNA4/ CRT-E2) were conrmed by immunouorescent assay (IFA). In the trial one, six-week-old BALB/c mice were intramuscularly injected four times with various doses of pcDNA4/E2 or plasmid vector at two-week intervals. Mice receiving 100 mg of pcDNA4/E2 showed signicantly higher (P<0.1) anti-E2 ELISA titers and signicantly higher (P<0.05) stimulation index (SI) of splenocytes proliferative response with stimulations of Con A or 20 mg of CSFV E2 protein than those of mice receiving plasmid vector only. The results of trial one showed that DNA vaccine containing CSFV E2 gene was able to induce humoral and cellular immune responses in mouse model. In the trial two, mice were vaccinated with vector, pCDNA4/CRT, pcDNA4/E2 or pcDNA4/CRT-E2 three times at two-week intervals by intramuscularly injection or gene gun. Mice receiving pcDNA4/E2 and pcDNA4/CRT-E2 showed signicantly higher (P<0.05) anti-E2 ELISA titers and signicantly higher (P<0.05) SI of splenocytes proliferative response with stimulation of 20 mg of CSFV E2 protein than those of mice receiving plasmid vector or pcDNA4/CRT only. Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
171. Generation of Chimeric GB Virus B Clones Encoding the Hepatitis C Virus E1E2p7 and coreE1E2p7 toward the Infected Marmoset Models
Tingting Li,1 Lifang Shuai,1 Zixuan Chen,1 Anqi Wang,1 Wenjing Wang,1 Chengyao Li.1 1 School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China.
The development of novel therapy and vaccination for hepatitis C virus (HCV) has been hampered by a lack of a small primate model. GB virus B (GBV-B) is closely related to HCV that causes acute or chronic hepatitis in marmosets, and thus, is an attractive surrogate model for HCV. Even more attractive, for mostly mimicking HCV infection in marmoset models, is the idea of constructing chimeric viruses by replacing GBV-B elements with its HCV counterpart of interest. Two infectious GBV-B chimeric genomes, HCV E1E2p7/ GBV-B and HCV coreE1E2p7/GBV-B, containing the coding region of E1 E2 p7 proteins and core E1 E2 p7 proteins were constructed, respectively. Overlapping PCR and restriction-enzyme digestion methods were used to generate the chimeric plasmids and then RNA transcripts were synthesized in vitro. The chimera may prove useful for establishing HCV infected marmoset models and determining HCV epitopes and immune responses in vivo. S66
172. Characterization of Immune Response in Mice Induced by Recombinant Vaccinia Virus (Tiantan) Based Multivalent H5N1 Avian Inuenza Vaccines
Wen Wang, Yao Deng, Wenjie Tan, Hong Chen, Li Ruan, Yuelong Shu. National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China. To develop an effective and broad immune protective H5N1 vaccine, We rst developed two recombinant vaccinia(Tiantan strain) virus(rTTV) based H5N1 vaccines, which consisted of bicistron expressing the hemagglutinin(HA) and matrix protein 2(M2),or bicistron expressing the neuraminidase(NA) and matrix protein 1(M1). The expression of H5N1 protein in rTTVs was conrmed. We immunized the Balb/C mice twice with two kind of dose (104 pfu, 107 pfu) using different combination. Subsequently, we assessed the humoral and cellular immune response in vaccinated mice. Our data showed that rTTV-based H5N1 vaccine induced rapidly robust HAand NA-specic antibody level and IFNγ secreting form cell(SFC) with either single dose of 107 pfu or twice dose of 104 pfu or 107 pfu. We also detected signicant neutralizing antibody and matrix-specic immune response. In addition, we found that immunization with two kind of rTTV-based H5N1 vaccines induced much high level of matrix 2-specic antibody than that with single of rTTV-based H5N1 vaccine. In conclusion, rTTV-based H5N1 vaccines elicit board array of immunity and our study offers a promising alternative H5N1 prevaccine candidates with favorable potential to prevent various H5N1 pandemic.
173. The Effects of the Calreticulin on the Immune Response Induced by DNA Vaccine Containing the Classical Swine Fever Virus E2 Gene in Mouse Model
Ming Kun Hsieh, Chia Chin You, Chienjin Huang. Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan. The structural glycoprotein E2 of the Classical swine fever virus (CSFV) can stimulate the production of neutralizing antibody which can protect pigs against CSF. Previous reports showed that DNA vaccine containing the E2 gene of CSFV was able to provide protective immunity. Calreticulin (CRT) has demonstrated the ability to enhance MHC class I presentation and elicit antigen-specic CD8+ T cell immune responses. The purpose of this study was to determine whether the mouse CRT gene can enhance the immune response of DNA vaccine by fusing with the E2 gene of CSFV in mouse model. Protein expression of plasmids containing CSFV E2 gene (pcDNA4/E2), mouse CRT gene (pcDNA4/CRT) or both (pcDNA4/ CRT-E2) were conrmed by immunouorescent assay (IFA). In the trial one, six-week-old BALB/c mice were intramuscularly injected four times with various doses of pcDNA4/E2 or plasmid vector at two-week intervals. Mice receiving 100 mg of pcDNA4/E2 showed signicantly higher (P<0.1) anti-E2 ELISA titers and signicantly higher (P<0.05) stimulation index (SI) of splenocytes proliferative response with stimulations of Con A or 20 mg of CSFV E2 protein than those of mice receiving plasmid vector only. The results of trial one showed that DNA vaccine containing CSFV E2 gene was able to induce humoral and cellular immune responses in mouse model. In the trial two, mice were vaccinated with vector, pCDNA4/CRT, pcDNA4/E2 or pcDNA4/CRT-E2 three times at two-week intervals by intramuscularly injection or gene gun. Mice receiving pcDNA4/E2 and pcDNA4/CRT-E2 showed signicantly higher (P<0.05) anti-E2 ELISA titers and signicantly higher (P<0.05) SI of splenocytes proliferative response with stimulation of 20 mg of CSFV E2 protein than those of mice receiving plasmid vector or pcDNA4/CRT only. Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy