Adjuvant Effects of Dual Co-stimulatory Molecules on Cellular Responses to HIV Multiple-epitope DNA Vaccination

Designing adjuvants that can induce strong cytotoxic T cell responses is largely required for preparing DNA vaccines. In this study we explored dual costimulatory molecules 4-1BBL and OX40L as adjuvants to improve the efficiency of the HIV multiple-epitope DNA vaccine. When explored in the human dendritic cell-T cell based coculture system, dual costimulatory molecules significantly enhanced the anti-HIV T cell response of the HIV multiple-epitope DNA vaccine, as detected by intracellular cytokine staining to HIV antigens, cytokines accumulation in the cultures, and antigen-specific cytotoxic T lymphocyte responses. These results suggest that dual costimulatory molecules 4-1BBL and OX40L can effectively increase the potential of the HIV multiple-epitope antigen DNA vaccine and may provide an exciting approach for HIV therapy.

Molecular Design and Immunogenicity of a Multiple-epitope FMDV Antigen and DNA Vaccination

This article reports the design and construction of a multiple-epitope foot and mouth disease virus （FMDV） antigen, designated as OAAT. This recombinant antigen consists of the structural protein VP1 genes from serotypes A and O FMDV, five major VP1 immunodominant epitopes from two genotypes of Asial serotype, and three Th2 epitopes originating from the nonstructural protein, three ABC gene and structural protein VP4 gene. Expressions of target gene from these plasmids in HeLa cells were verified by Western-blot. BALB/c mice were immunized intramuscularly with the DNA vaccines thrice every two weeks. We found that pA could induce simultaneously specific antibodies against serotypes A, Asial, and O FMDV. Compared to those of the controls, the spots of FMDV-specific IFN-7 and cytotoxic activity from mice immunized with pA were significantly increased, pA provided full protection in 2/4 guinea pigs from challenge with FMDV O/NY00 and Asial/YNBS/58, respectively. The results show that although pA did not give full protection in 100% immunized guinea pigs from challenge with type O and Asial FMDV, respectively, OAAT may be potential immunogen against FMDV and pA may be potential DNA vaccines against FMDV.

Vaccination of Plasmid DNA Encoding Somatostatin Gene Fused with GP5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Induces Anti-GP5 Antibodies and Promotes Growth Performance in Immunized Pigs

Somatostatin （SS） is a hormone that inhibits the secretion of growth hormone. Immunization against SS can promote the growth of animals. This paper described the effects of DNA immunization on the growth and antibody response in mice and pigs immunized with a plasmid DNA encoding SS fused with GP5 of porcine reproductive and respiratory syndrome virus （PRRSV）. A fragment of 180 bp encoding partial SS gene was amplified by PCR from the genomic DNA of peripheral blood mononuclear cells of pigs, and cloned as a fusion gene with PRRSV GP5 in plasmid pISGRTK3. Three times of immunization with the resulting plasmid pISG-SS/GP5 induced anti-GP5 antibodies in BALB/c mice and pigs, as demonstrated by GP5-specific ELISA and immunoblotting. Compared with pigs immunized with empty vector pISGRTK3, the growth performance of pigs immunized with pISG-SS/GP5 was increased by 11.1% on the 13th week after the last vaccination. The results indicated the plasmid DNA encoding SS and PRRSV GP5 fusion gene elicited anti-GP5 antibodies and improved the growth performance of immunized pigs.

Construction and Immunogenicity of Associated DNA Vaccine of PRRS and PCV-2 Disease

[ Objective ] The aim of the study was to construct associated DNA vaccine of PRRS （Porcine reproductive and respiratory syndrome） and PCV-2 （Porcine circovirus type 2） disease and study its immunogenicity. [ Method] In_ this study, the ORF5 gene of PRRSV isolated in Liaoning was cloned into plRES-neo expression vector, and the neo gene of plRES-neo expression vector was substituted by the ORF2 gene of the PCV-2 Mongolia strain to construct the recombinant expression vector. The expression in BHK cells was detected through Western blot and IFA. Then the ELISA antibody level and the number of spleen T lymphocytes were detected after Balb/c mice were immunized with this DNA vaccine. E Result] The recombinant plasmid plRES-ORF2-ORF5 was constructed successfully and could express the target proteins in BHK cells, as indicated by Western blot and IFA. There was no significant difference in ELISA antibody between plRES-ORF2-ORF5 immunized group and inactived vaccine immunized groups, while the number of spleen T lymphocytes induced by DNA vaccine was higher than that induced by inactived vaccine. [ Conclusion] The recombinant plasmid plRES-ORF2-ORF5 should induce good humoral immune response and cellular immune response in mice, providing the conditions for better prevention and control of PRRS and PCV-2 disease.

Immunogenicity and protective efficacy of Vibrio harveyi pcFlaA DNA vaccine in Epinephelus awoara

The FlaA gene from Vibrio harveyi marker, was cloned into the eukaryotic expression with a short nucleotide sequence encoding the Flag vector pcDNA3.1（＋） （designated as pcFlaA）. Ninety grouper （Epinephelus awoara） were separated into three equal size groups. An experimental group was immunized with pcFlaA, Control I group was immunized with the vector pcDNA3.1（＋）, and Control 1I group was immunized with PBS. The expression of pcFlaA mRNA and protein was examined using reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry. We also evaluated the immunogenicity and protective efficacy of pcFlaA against V. harveyi by measuring the lymphocyte proliferation response and serum levels of specific antibody and conducting a bacterial challenge test. We successfully transfected the fish muscle with pcFlaA. The pcFlaA mRNA and protein was expressed in the muscle cells for up to one month following injection. The proliferation response of lymphocytes in fish immunized with pcFlaA was significantly higher than in control group II. Furthermore, the immunized fish generated specific antibody. The vaccination also resulted in significantly higher survival during the bacterial challenge test.

Modulation of cellular and humoral immune responses to anHIV-1 DNA vaccine by interleukin-12 and interleukin-18 DNA immunization

Objective: To investigate the effect of interleukin-12 (IL-12) and interleukin-18 (IL-18)DNA immunization on immune response induced by HIV-1 DNA vaccine and to explore new strategies for therapeutic HIV DNA vaccine. Methods: The recombinant expression vector pCI-neoGAG was constructed by inserting HIV Gag gene into the eukaryotic expression vector pCI-neo. Balb/c mice were immunized with pCI-neoGAG alone or co-immunized with the DNA encoding for IL-12 or IL-18.Anti-HIV antibody and IFN-γ were tested by ELISA,and splenocytes were isolated for detecting antigen-specific lymphoproliferative responses and specific CTL response by MTT assay and LDH assay respectively. Results: The anti-HIV antibody titers of mice co-immunized with pCI-neoGAG and the DNA encoding for IL-12 or IL-18 were lower than that of mice immunized with pCI-neoGAG alone(P<0.01). In contrast, the IFN-γ level of mice co-immunized with pCI-neoGAG and the DNA encoding for IL-12 or IL-18 was higher than that of mice immunized with pCI-neoGAG alone (P<0.01).Furthermore, compared with mice injected with pCI-neoGAG alone, the specific CTL cytotoxity activity and antigen-specific lymphoproliferative responses of mice immunized with pCI-neoGAG and the DNA encoding for IL-12 or IL-18 were significantly enhanced respectively (P<0.01). Conclusion: The DNA encoding for IL-12 or IL-18 together with HIV DNA vaccine may enhance specific Th-1 responses and cellular immune response elicited in mice. Hence, the DNA encoding for IL-12 or IL-18 are promising immune adjuvants for HIV-1 DNA vaccine.

Construction of recombinant attenuated Salmonella typhimurium DNA vaccine expressing H pylori ureB and IL-2

AIM： To construct a recombinant live attenuated Salmonella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS： Hpylori ureB and mouse IL-2 gene fragments were amplified by polymerase chain reaction （PCR） and cloned into pUCmT vector. DNA sequence of the amplified ureB and IL-2 genes was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions resulting in pIRES-ureB and pIRES-ureB-IL-2. The recombinant plasmids were used to transform competent E. co/i DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-ureB and pIRES-ureB-IL-2 were used to transform LB5000 and the recombinant plasmids extracted from LB5000 were finally introduced into the final host SL7207. After that, recombinant strains were grown in vitro repeatedly. In order to detect the immunogenicib/of the vaccine in vitro, pIRES-ureB and pIRES-ureB-IL-2 were transfected to COS-7 cells using LipofectamineTM2000, the immunogenicity of expressed UreB and IL-2 proteins was assayed with SDS-PAGE and Western blot. C57BL/6 mice were orally immunized with 1 × 10^8 recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 × 10^7 CFU of live Hpylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylon 4 wk post-challenge. RESULTS： The 1700 base pair ureB gene fragment amplified from the genomic DNA was consistent with the sequence of H pylori ureB by sequence analysis. The amplified 510 base pair fragment was consistent with the sequence of mouse IL-2 in gene bank. It was confirmed by PCR and restriction enzyme digestion that H pylori ureB and mouse IL-2 genes were inserted into the eukaryotic expression vector pIRES. The experiments in vitro showed that stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying ureB and IL-2 genes was successfully constructed and the specific strips of UreB and IL-2 expressed by recombinant plasmids were detected through Western blot. Study in vivo showed that the positive rate of rapid urease test of the immunized group including ureB and ureB-IL-2 was 37.5% and 12.5% respectively, and was significantly lower than that （100%） in the control group （P 〈 0.01）. CONCLUSION： Recombinant attenuated Salmonella typhimurium DNA vaccine expressing UreB protein and IL-2 protein with immunogenicity can be constructed. It can protect mice against H pylori infection, which may help the development of a human-use H pylori DNA vaccine.

Novel DNA vaccine based on hepatitis B virus core gene induces specific immune responses in Balb/c mice

AIM： To investigate the immunogenidty of a novel DNA vacoine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS： A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes （CTLs） of mice was quantitatively measured by lactate dehydrogenase release assay. RESULTS： HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1：97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector：target ratio of 20：1 in mice that received pSW3891/HBc DNA vaccine. CONCLUSION： pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.

Targeting hepatitis B virus antigens to dendritic cells by heat shock protein to improve DNA vaccine potency

AIM： To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein （HSP） as a strategy to enhance DNA vaccine potency.METHODS： A pCMV-HBeAg-HSP DNA vaccine and a control DNA vaccine were generated. Mice were immunized with these different construct. Immune responses were measured 2 wk after a second immunization by a T cell response assay, CTL cytotoxicity assay, and an antibody assay in C57BL/6 and BALB/c mice. CT26-HBeAg tumor cell challenge test in vivo was Performed in BALB/c mice to monitor anti-tumor immune responses.RESULTS： In the mice immunized with pCMV-HBe-HSP DNA, superior CTL activity to target HBV-positive target cells was observed in comparison with mice immunized with pCMV-HBeAg （44% ± 5% vs 30% ± 6% in E： T 〉 50：1, P 〈 0,05）, ELISPOT assays showed a stronger T-cell response from mice immunized with pCMV-HBe- HSP than that from pCMV-HBeAg immunized animals when stimulated either with MHC class I or class Ⅱ epitopes derived from HBeAg （74% ± 9% vs 31% ± 6%, P 〈 0.01）. ELISA assays revealed an enhanced HBeAg antibody response from mice immunized with pCMV- HBe-HSP than from those immunized with pCMV-HBeAg. The lowest tumor incidence and the slowest tumor growth were observed in mice immunized with pCMV- HBe-HSP when challenged with CT26-HBeAg.CONCLUSION： The results of this study demonstrate a broad enhancement of antigen-specific CD4^＋ helper,CD8^＋ cytotoxic T-cell, and B-cell responses by a novel DNA vaccination strategy. They also proved a stronger antigen-specific immune memory, which may be superior to currently described HBV DNA vaccination strategies for the treatment of chronic HBV infection.

Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant

Objective:To investigate the effect of interleukin-18 (IL-18) on immune response induced by plasmid encoding hepatitis B virus middle protein antigen and to explore new strategies for prophylactic and therapeutic HBV DNA vaccines.Methods:BALB/c mice were immunized with pCMV-M alone or co-immunized with pcDNA3-18 and pCMV-M and then their sera were collected for analysing anti-HBsAg antibody by ELISA;splenocytes were isolated for detecting specific CTL response and cytokine assay in vitro.Results:The anti-HBs antibody level of mice co-immunized with pcDNA3-18 and pCMV-M was slightly higher than that of mice immunized with pCMV-M alone,but there was not significantly different (P>0.05).Compared with mice injected with pCMV-M, the specific CTL cytotoxity activity of mice immunized with pcDNA3-18 and pCMV-M was significantly enhanced (P<0.05) and the level of IFN-γ in supernatant of splenocytes cultured with HBsAg in vitro was significantly elevated (P<0.05) while the level of IL-4 had no significant difference (P>0.05).Conclusion:The plasmid encoding IL-18 together with HBV M gene DNA vaccines may enhance specific TH1 cells and CTL cellular immune response induced in mice, so that IL-18 is a promising immune adjuvant.

IFN-γ increases efficiency of DNA vaccine in protecting ducks against infection

AIM： To detect the effects of DNA vaccines in combination with duck IFN-γ gene on the protection of ducks against duck hepatitis B virus （DHBV） infection. METHODS： DuIFN-γ cDNA was cloned and expressed in COS-γ cells, and the antiviral activity of DuIFN-γ was detected and neutralized by specific antibodies, Ducks were vaccinated with DHBpreS/S DNA alone or coimmunized with plasmid expressing DuIFN-γ. DuIFN-γ mRNA in peripheral blood mononuclear cells （PBMCs） from immunized ducks was detected by semi-quantitative competitive RT-PCR. Anti-DHBpreS was titrated by enzyme-linked immunosorbent assay （EUSA）. DHBV DNA in sera and liver was detected by Southern blot hybridization, after ducks were challenged with high doses of DHBV. RESULTS： DuIFN-γ expressed by COS-γ was able to protect duck fibroblasts against vesicular stomatitis virus （VSV） infection in a dose-dependent fashion, and anti DuIFN-γ antibodies neutralized the antiviral effects. DuIFN-γ in the supernatant also inhibited the release of DHBV DNA from LMH-D2 cells. When ducks were co-immunized with DNA vaccine expressing DHBpreS/S and DuIFN-γ gene as an adjuvant, the level of DuIFN-γ mRNA in PBMCs was higher than that in ducks vaccinated with DHBpreS/S DNA alone. However, the titer of anti-DHBpreS elicited by DHBpreS/S DNA alone was higher than that co-immunized with DuIFN-γ gene and DHBpreS/S DNA. After being challenged with DHBV at high doses, the load of DHBV in sera dropped faster, and the amount of total DNA and cccDNA in the liver decreased more significantly in the group of ducks co-immunized with DuIFN-γ gene and DHBpreS/S DNA than in other groups.

Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylorihpaA

AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. METHODS: Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then doned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coliDH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine^(TM)2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. RESULTS: The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. CONCLUSION: The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.

Interleukin-12 as a Genetic Adjuvant Enhances Hepatitis C Virus NS3 DNA Vaccine Immunogenicity

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.

Coimmunization with IL-15 plasmid enhances the longevity of CD8 T cells induced by DNA encoding hepatitis B virus core antigen

AIM： To test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for improving the immune responses induced by hepatitis B virus core gene DNA vaccine. METHODS： We used RT-PCR based strategies to develop IL-15 expression constructs. We first confirmed that the gene could be expressed in Escherichia coli due to the poor expression of IL-15. Then the bioactivity of IL-15 plasmid expression product was identified by CTLL-2 proliferation assay. One hundred micrograms of DNA from each of the IL-15 eukaryotic expressed plasmid and the recombinant plasmid harboring DNA encoding the 144 amino acids of the N-terminus of HBV core gene （abbreviated pHBc144） was used to co-immunize C57 BL/6 mice. The titer of anti-HBcIgG was detected by ELISA and the antigen-specific CD8^＋T cells （CD8^＋IFN-γ^＋ T cells） were detected by intracellular cytokine staining at different time points. RESULTS： After co-immunization by pIL-15 and pHBc144 DNA vaccine the antigen-specific CD8^＋ cells of mice increased gradually, the first peak of immune response appeared 14 d later, then the number of antigen-specific CD8^＋Ts cells decreased gradually and maintained at a steady level in 3 mo. After boosting, the number of antigen-specific CD8^＋T cells reached the second peak 10 d later with a double of the 1st peak, then the number of antigen-specific CD8^＋T cells decreased slowly. IL-15 as a gene adjuvant had no significant effect on humoral immune responses induced by hepatitis B virus core gene DNA vaccine, but increased the memory antigen-specific CD8^＋T cells induced by hepatitis B virus core gene DNA vaccine. CONCLUSION： DNA vaccine constructed by HBc Ag 1-144 amino acid induces effective cell immunity, and cytokine plasmid-delivered IL-15 enhances the longevity of CD8^＋ T cells.

Recent advances in DNA vaccine of hepatitis virus

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.

Immune Responses in Wild-type Mice Against Prion Proteins Induced Using a DNA Prime-Protein Boost Strategy

Objective To break immune tolerance to prion （PrP） proteins using DNA vaccines.Methods Four different human prion DNA vaccine candidates were constructed based on the pcDNA3.1 vector：PrP‐WT expressing wild‐type PrP,Ubiq‐PrP expressing PrP fused to ubiquitin,PrP‐LII expressing PrP fused to the lysosomal integral membrane protein type II lysosome‐targeting signal,and PrP‐ER expressing PrP locating the ER.Using a prime‐boost strategy,three‐doses of DNA vaccine were injected intramuscularly into Balb/c mice,followed by two doses of PrP protein.Two weeks after the last immunization,sera and spleens were collected and PrP‐specific humoral and cellular immune responses evaluated by ELISA and ELISPOT tests.Results Higher levels of serum PrP antibodies were detected in mice vaccinated using the strategy of DNA priming followed by protein boosting.Of these,WT‐PrP,Ubiq‐PrP,and PrP‐LII induced significantly higher humoral responses.ELISPOT tests showed markedly increased numbers of IFN‐γ‐secreting T cells in mice vaccinated using the strategy of DNA priming followed by protein boosting after stimulation with recombinant PrP23‐90 and PrP23‐231.PrP‐ER inducedthe strongest T‐cell response.Conclusion Prion vaccines can break tolerance to PrP proteins and induce PrP‐specific humoral and cellular immune responses.

Phase Ⅱb trial of in vivo electroporation mediated dualplasmid hepatitis B virus DNA vaccine in chronic hepatitis B patients under lamivudine therapy

AIM To assess the efficacy and safety of in vivo electroporation(EP)-mediated dual-plasmid hepatitis B virus(HBV) DNA vaccine vs placebo for sequential combination therapy with lamivudine(LAM) in patients with chronic hepatitis B. METHODS Two hundred and twenty-five patients were randomized to receive either LAM + vaccine(vaccine group, n = 109) or LAM + placebo(control group, n = 116). LAM treatment lasted 72 wk. Patients received the DNA vaccine or placebo by intramuscular injection mediated by EP at weeks 12(start of treatment with vaccine or placebo, SOT), 16, 24, and 36(end of treatment with vaccine or placebo, EOT). RESULTS In the modified intent-to-treat population, morepatients had a decrease in HBV DNA > 2 log10 IU/m L in the vaccine group at week 12 after EOT compared with the control group. A trend toward a difference in the number of patients with undetectable HBV DNA at week 28 after EOT was obtained. Adverse events were similar. In the dynamic per-protocol set, which excluded adefovir(ADV) add-on cases at each time point instantly after ADV administration due to LAM antiviral failure, more patients had a decrease in HBV DNA > 2 log10 IU/mL in the vaccine group at week 12 and 28 after EOT compared with the control group. More patients with undetectable HBV DNA at week 28 after EOT in the vaccine group were also observed. Among patients with a viral load < 1000 copies/mL at week 12, more patients achieved HBeA g seroconversion in the vaccine group than among controls at week 36 after EOT, as well as less virological breakthrough and YMDD mutations. CONCLUSION The primary endpoint was not achieved using the HBV DNA vaccine. The HBV DNA vaccine could only be beneficial in subjects that have achieved initial virological response under LAM chemotherapy.

Construction and Expression of DNA Vaccine pIRES-Sj97-Sj14-Sj26 and Its Immunogenicity in Mice

To find a new preventive strategy for the infection of Schistosoma japonica, plasmid pIRES-Sj97-Sj 14-Sj26 that contains fatty binding protein （Sj 14）, GST （Sj26） and paramyocin （Sj97） that are expressed on the membrane, was constructed. RT-PCR was used to detect the expression of Sj 14 mRNA, Sj26 mRNA and Sj97 mRNA in the Hela cells, the indirect immunofluorescent test was employed for the detection of the expression of trans-membrane Sj26 after the plasmid was transfected into Hela cells. Fifty BALB/c mice were randomly divided into 5 groups and plRES-Sj97-SjI4-Sj26 plasmid DNA, plRES-Sj 14-Sj26 plasmid DNA, plRES-Sj26 plasmid DNA, plRES blank vector and normal saline were respectively injected into the quadriceps muscles of thigh Eight weeks after the immunization the mice were killed and significantly higher level of IgG was detected in the plRES-Sj97-Sj 14-Sj26 group as compared with the plRES blank vector, normal saline and plRES-Sj26 groups （P〈 0.01） and the plRES-Sj 14-Sj26（P〈0.05）. Single splenocyte suspension was prepared to detected the level of IFN-T by ELISA and the lymphocyte stimulating index （SI） by MTT. SI was significantly higher of in the plRES-Sj97-Sj 14-Sj26 group than in other groups （P〈 0.01）, while the IFN-T level was significantly higher the plRES-Sj97-Sj 14-Sj26 group than in plRES blank vector and normal saline groups （P〈0.01）, but no significant differences were found when compared with plRES-Sj 14-Sj26 and plRES-Sj26 groups. Flow cytometery showed that the percentages of CD4＋ and CD8＋ T cells were much higher in the plRES-Sj97-Sj 14-Sj26 group （P〈 0.01, P〈0.05）. It was concluded that plRES-Sj97-Sj 14-Sj26 vaccine may induce stronger immune response in BALB/c mice.

In Vivo Kinetics and Biodistribution of a Hantaan Virus DNA Vaccine after Intramuscular Injection in Mice

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.

Comparison of Immune Responses against FMD by a DNA Vaccine Encoding the FMDV/O/IRN/2007 VP1 Gene and the Conventional Inactivated Vaccine in an Animal Model

Foot-and-mouth disease virus （FMDV） is highly contagious and responsible for huge outbreaks among cloven hoofed animals. The aim of the present study is to evaluate a plasmid DNA immunization system that expresses the FMDV/OflRN/2007 VP1 gene and compare it with the conventional inactivated vaccine in an animal model. The VP1 gene was sub-cloned into the unique Kpn I and BamH I cloning sites of the peDNA3.1＋ and pEGFP-N1 vectors to construct the VPI gene cassettes. The transfected BHKT7 cells with sub-cloned pEGFP-N1-VP1 vector expressed GFP-VP1 fusion protein and displayed more green fluorescence spots than the transfected BHKT7 cells with pEGFP-N1 vector, which solely expressed the GFP protein. Six mice groups were respectively immunized by the sub-cloned pcDNA3.1＋-VP1 gene cassette as the DNA vaccine, DNA vaccine and PCMV-SPORT-GMCSF vector （as molecular adjuvant） together, conventional vaccine, PBS （as negative control）, pcDNA3.1＋ vector （as control group） and PCMV-SPORT vector that contained the GMCSF gene （as control group）. Significant neutralizing antibody responses were induced in the mice which were immunized using plasmid vectors expressing the VP1 and GMCSF genes together, the DNA vaccine alone and the conventional inactivated vaccine （P〈0.05）. Co-administration of DNA vaccine and GMCSF gene improved neutralizing antibody response in comparison with administration of the DNA vaccine alone, but this response was the most for the conventional vaccine group. However, induction of humeral immunity response in the conventional vaccine group was more protective than for the DNA vaccine, but T-cell proliferation and IFN-？ concentration were the most in DNA vaccine with the GMCSF gene. Therefore the group that was vaccinated by DNA vaccine with the GMCSF gene, showed protective neutralizing antibody response and the most Thl cellular immunity.