Humoral and cellular immunogenecity of DNA vaccine based on hepatitis B core gene in rhesus monkeys

INTRODUCTIONHepatitis B virus (HBV) is the most commonetiologic agent for infectious liver diseases. It isestimated that there are more than 250 millionchronic HBV carriersin the world today and thereis a significant association among persistentinfection, liver cirrhosis and hepatocellularcarcinoma[1-3].

The past, current and future trends in DNA vaccine immunisations

This review focuses on DNA vaccines, denoting the last two decades since the early substantiation of preclinical protection was published in Science in 1993 by Ulmer et al. In spite of being safely administered and easily engineered and manufactured DNA vaccine, it holds the future prospects of immunization by inducing potent cellular immune responses against infectious and non-infectious diseases. It is well documented that injection of DNA plasmid encoding a desired gene of interest can result in the subsequent expression of its products and lead to the induction of an immune response within a host. This is pertinent to prophylactic and therapeutic vaccination approach when the peculiar gene produces a protective epitope from a pathogen. The recent studies demonstrated by a number of research centers showed that these immune responses evoke protective immunity against several infectious diseases and cancers, which provides adequate support for the use of this approach. We attempt in this review to provide an informative and unbiased overview of the general principles and concept of DNA vaccines technology with a summary of a novel approach to the DNA vaccine, present investigations that describe the mechanism(s) of protective immunity provoked by DNA immunization and to highlight the advantages and disadvantages of DNA immunisation.

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.

铜绿假单胞菌oprI基因DNA疫苗及其重组亚单位疫苗免疫效果的评估

为评估铜绿假单胞菌opr I基因DNA疫苗和重组亚单位疫苗对小鼠的免疫效果,本实验将铜绿假单胞菌外膜蛋白编码基因opr I克隆至真核表达载体p CAGGS-HA中构建重组质粒p CAGGS-opr I,经酶切鉴定正确后获得DNA疫苗。同时将opr I基因克隆至原核表达载体p ET32a中构建重组质粒p ET32a-oprI,经酶切鉴定正确后转入大肠杆菌,经IPTG诱导重组Opr I蛋白(rOprI)的表达,采用亲和层析法纯化后以SDS-PAGE检测,结果显示正确表达了r Opr I,且获得了其纯化蛋白,利用其制备重组亚单位疫苗。分别以DNA疫苗和重组亚单位疫苗免疫BALB/c小鼠,并以铜绿假单胞菌的灭活疫苗和外膜蛋白疫苗为对照,采用间接ELISA法检测免疫后不同时间小鼠血清中的抗体水平和血清中IFN-γ、IL-2和IL-4的含量;初免42 d后以铜绿假单胞菌(1.25×109 cfu/mL,100μL/只)对各组小鼠进行攻毒试验,通过测定各疫苗对小鼠的保护率,评估疫苗的保护效果。结果显示,各疫苗诱导的免疫小鼠血清抗体水平和细胞因子含量均显著高于阴性对照组(P<0.05),且重组亚单位疫苗诱导小鼠的抗体水平和IL-4含量均显著高于DNA疫苗(P<0.05),而IFN-γ和IL-2含量则与DNA疫苗无显著差异(P>0.05)。小鼠攻毒试验结果显示,DNA疫苗组、重组亚单位疫苗组、外膜蛋白疫苗组和灭活疫苗组小鼠获得的免疫保护率分别为45%、55%、70%和95%。上述结果首次表明以铜绿假单胞菌opr I基因制备的DNA疫苗和r Opr I重组亚单位疫苗均可诱导小鼠产生体液和细胞免疫应答,并可为小鼠提供对铜绿假单胞菌攻毒的免疫保护效果,但二者的免疫效果还需进一步提高。本研究为铜绿假单胞菌疫苗的研究提供了参考依据。

Protective effect of DNA vaccine with the gene encoding 55kDa antigen fragment against Pneumocystis carinii in mice

Objective:To evaluate the protective effect of DNA vaccine with the gene encoding 55kDa antigen fragment of Pneumocystis carinii(P.carina) against P.carina in mice.Methods:The fragment of the antigen within p55(p55-582) was cloned.Then recombinant plasmid was constructed based on the eukaryotic expression vector pcDNA3.1(+).BALB/c mice were used as experimental models to examine the immunogenicity of pcDNA3.1(+)-p55-582.ELBA and RTPCR were used to evaluate the role of this kind of DNA vaccine.Results:The results of western blot indicated that the recombinant DNA[pcDNA3.1(+)-p55-582]could be expressed correctly and had antigenicity in transfected COS-7 cells.ELBA and RT-PCR showed that pcDNA3.1(+)- p55-582 elicited antibody production,stimulated lymphocyte proliferation and provided partial protection by reducing the P.carina burden.Conclusions:The data demonstrate that pcDNA3.1(+)-p55-582 might be potent vaccination that can afford the partial protection for the immunized animals.

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.

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.

Specific cellular immunity and antitumor responses in C57BL/6 mice induced by DNA vaccine encoding murine AFP

BACKGROUND: The inoculation of plasmid DNA encod-ing tumor-associated antigens is a novel and powerful stra-tegy for antitumor vaccination. This study was designed toconstruct the DNA vaccine of mouse AFP and to observethe specific cellular immunologic responses and the antitu-mor responses in mice induced by this vaccine.METHODS: The murine AFP gene was amplified by RT-PCR from total RNA extracted from Hepal-6 cells andcloned into the vector pcDNA3.1 to construct pmAFP.The DNA vaccine was identified by restriction enzymeanalysis, sequencing and expression. EL-4 ( mAFP) wasdeveloped by stable transfection of EL-4 cells with pmAFP.The frequency of cells producing IFN-γ in splenocytes har-vested from the mice immunized with the DNA vaccine byintramuscular injection was measured by enzyme linkedimmunospot (ELISPOT). The mice immunized with theDNA vaccine were inoculated with EL-4 (mAFP) cells inback to observe the inhibitory effect of the immunizationon tumor. On the other hand, blood samples were collect-ed from the immunized mice to check the functions of theliver and kidney.RESULTS: The murine AFP gene was successfully clonedby RT-PCR. Results from restriction enzyme analysis, se-quencing and expression showed that the DNA vaccine wassuccessfully constructed. The expression of mAFP mRNAin EL-4 (mAFP) was confirmed by RT-PCR. The resultsof ELISPOT showed that the number of IFN-γ-producingcells of the pmAFP vaccine group was significantly higherthan that of other groups (P <0.01). The tumor volume inthe pmAFP vaccine group (1042. 42 ± 123. 71 mm3 ) wassignificantly smaller than that in other groups (P<0.01).The function of mouse liver and kidney in each group wasunchanged.CONCLUSION: The successfully constructed DNA vaccineof AFP can induce specific cellular immunologic responsesand significant antitumor reponses in mouse and has no im-pact on the function of mouse liver and kindey.

Immunogenicity and immunizing protection effect of GAMA gene DNA vaccine on Plasmodium berghei

Objective: To explore the effect of immunogenicity and immunizing protection of GAMA gene DNA vaccine, which was related with merozoite, ookinete and sporozoite invasion. Methods:Gene fragments were obtained using PCR technique and eukaryotic expression vector(containing immunostimulatory sequence) was built. BALB/c mice were divided into PBS control group, empty vector control group and study group and were immunized at week 0, 3 and 6 respectively. Blood was collected 2 weeks after each immunization and serum was separated to detect the Ig G, Ig G1 and Ig G2 a levels. Spleen of mice was obtained for preparation of splenic mononuclear cell and the cytokine IL-4 and IFN-αlevels were detected. Indirect immunofluorescence and western blot were employed to verify the specificity of antiserum. Sporozoite and merozoite invasion were used respectively to detect the immune protective effect 2 weeks after the third immunization. Ookinete conversion rate in vitro and oocyst numbers of mosquito stomach were observed to evaluate the transmission-blocking levels. Results: In GAMA DNA vaccine group: antiserum could be combined with recombinant protein specifically and green fluorescence signals of merozoite, ookinete and sporozoite were observable, while specific fragments and fluorescence signals were not observable in empty vector group. Compared with control group, specific Ig G in DNA vaccine immunity group significantly increased(P<0.01), and Ig G1 and Ig G2 a all increased(P<0.01). IL-4, IFN-αcontent in study group significantly increased, compared with control group(P<0.01). GAMA DNA vaccine immunity could not obviously block the erythrocyte-stage infection(caused by sporozoite invasion); compared with control group, liver worm load was slightly reduced(P<0.05), and antiserum ookinete numbers(cultured in vitro) had no significant difference with oocyst numbers of mosquito stomach in DNA vaccine group. Conclusions:GAMA has good antigenicity, which could stimulate the body to produce specific immune responses; while DNA vaccine immunity could not play a good protective effect, the effect of which is only limited to the slight reduction of liver worm load, and has no obvious erythrocytestage protective effect and transmission-blocking effect. Therefore, trying other immunization strategies for further research on the value of GAMA(as multi-stage antigen vaccine and multistage combined vaccine components of the life-cycle of plasmodium) is necessary.

The Protection Efficacity of DNA Vaccine Encoding Hemagglutinin of H5 Subtype Avian Influenza Virus

The DNA vaccine pCIHA5 encoding hemagglutinin can protect SPF chicken against lethal H5N1 avian influenza virus challenge. The more characters about its protection efficacity were studied. The protective rates in 10, 40, 70, 100 and 150 μg groups immunized with pCIHA5 were 12.5 (1/8), 58.3 (7/12), 72.7 (8/11), 50.0 (6/12) and 66.7% (8/12), respectively. The protective rates in 5, 20, 35 and 50 μg groups were 145.5 (5/11), 58.3 (7/12), 58.3 (7/12) and 91.7% (11/12), respectively. The 70, 100 and 5 μg groups have virus shedding of 1/8, 2/6 and 1/5. Though the inactived oil-emulsion vaccine has high HI antibody titers and 100% protective rate, the AGP antibody could be detected after vaccination. Results show that the pCIHA5 is fit to boost by intramuscular injection. This would be useful to the study on gene engineering vaccine of avian influenza virus.

Studies on DNA Vaccine of the Conservative Region of Gene Encoding the Male-Specific Gynecophoral Canal Protein of Schistosoma japonicum (Chinese strain)

In order to observe the protective effect induced by vaccinating animals with the DNA vaccine of Sex-specific expression gene of Schistosoma, A 868 bp cDNA fragment amplified by RT-PCR from adult Schistosoma japonicum (Chinese strain) mRNA was cloned into the eukaryotic expression vector pcDNA3 and the recombinant eukaryotic expression vector pcDNA3-SjGCP1 was directly injected into BALB/c mice intramuscularly 3 times with the interval of 3 weeks .Both the vaccinated and control group of mice were challenged with 40 cercariae of Sj 5 weeks after last injection and perfused 7 weeks post-challenge. The worm and egg reduction rate got from vaccinated mice was 32.4% and 46.9% respectively. The result indicated that pcDNA3-SjGCP1 DNA vaccine induces the significant protection in animal against Schistosoma japonicum infection.

HPV16E7-HSP70 Hybrid DNA Vaccine Induces E7-Specific Cytotoxic T Cells and Antitumor Immunity

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.

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

AIM: To construct a recombinant live attenuated Salm-onella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS: H pylori 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. coli 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 immunogenicity 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 × 108 recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 × 107 CFU of live H pylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylori 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 consistentwith 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.

Comparative Study on the Immunogenicity between Hsp70 DNA Vaccine and Hsp65 DNA Vaccine in Human Mycobacterium Tuberculosis

The BALB/c mice were immunized with Hsp70 DNA and Hsp65 DNA vaccines in human Mycobacterium tuberculosis. Eight weeks after immunization, the eyeballs were removed, blood and spleen taken, and intraperitoneal macrophages were harvested. The lymphocytic stimulating index (SI) was used to measure the cellular proliferating ability and NO release to measure the phagocytic activity of the macrophages. With ELISA kit, the levels of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in serum and the splenic lymphocytic cultured supernatant were detected. The results showed that after the mice were immunized with 100 μg/mouse of Hsp70 DNA vaccine intramuscularly, the splenic lymphocytic proliferating ability in the mice was significantly increased as compared with that in the control group, vector group and Hsp65 DNA vaccine group (P<0.01); The contents of NO in the intraperitoneal macrophages of the mice were significantly lower than in the control group and Hsp65 DNA vaccine group (P<0.01); The levels of serum IL-2 in the mice were significantly higher than in the control group, but there was no statistical difference between Hsp65 DNA group and vector group (P>0.05); The contents of serum IFN-γ in the mice were significantly higher than in the control group, but significantly lower than in the Hsp65 DNA vaccine group (P<0.05). It was indicated that immunization with Hsp70 DNA vaccine could obviously enhance the immune response, but its intensity seemed inferior to Hsp65 DNA vaccine. The anti-infection mechanisms and clinical use in the future of the vaccines of Hsp70 DNA and Hsp65 DNA are worth further studying.

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 cloned 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 vitrorepeatedly. In order to iclentify the immunogenicity of the vaccinein vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using LipofectamineTM2000, 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.

Human Enterovirus 71 DNA Vaccine Constructs Containing 5’UTR with Complete Internal Ribosome Entry Site Sequence Stimulated Improved Anti-Human Enterovirus 71 Neutralizing Immune Responses

Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.

HPV16 E6、E7多表位DNA疫苗的构建及免疫效果评估

目的构建和评价HPV16 E6、E7多表位DNA疫苗诱导的特异性CTL细胞应答及其对肿瘤生长的干预作用,从而揭示其作为候选HPV治疗性疫苗的潜能。方法首先通过IEDB网站中的MHC I Processing Predictions和MHC I Binding Predictions方法,分别预测人类HLA-A^(*)02:01、HLA-A^(*)11:01、HLA-A^(*)24:02和C57BL/6小鼠H-2b的限制性CTL表位,然后根据评分以及ELISPOT实验筛选出二者共同呈递的CTL表位,并将其构建成多表位DNA疫苗(pVAX1-10P)。从预防性和治疗性二个方面研究pVAX1-10P对小鼠移植TC-1异位癌的免疫干预作用,流式细胞术检测特异性CTL应答。结果获得10条可被人与鼠MHC分子共呈递的CTL表位,ELISPOT结果表明这10条CTL表位均能诱导小鼠淋巴细胞产生特异性免疫应答;由此构建的多表位DNA疫苗pVAX1-10P无论在预防性实验还是治疗性实验中,均能诱导特异性的细胞免疫并抑制肿瘤的生长。结论构建的HPV16 E6、E7多表位DNA疫苗pVAX1-10P能够诱导特异性CTL应答,显著抑制肿瘤生长,有望作为候选HPV治疗性DNA疫苗。

Co-administration of Interleukin-2 Enhances Cellular and Humoral Immune Responses to HIV Vaccine DNA Prime/MVA Boost Regime

Interleukine-2(IL-2) is a growth factor for antigen-stimulated T lymphocytes and is responsible for ~T-cell clonal expansion after antigen recognition. It has been demonstrated that DNA vaccine-elicited immune responses in mice could be augmented substantially by using either an IL-2 protein or a plasmid expressing ~IL-2. Twenty mice, divided into four experimental groups, were immunized with: (1) sham plasmid; ^(2) HIV-1 DNA vaccine alone; (3) HIV-1 DNA vaccine and IL-2 protein; or (4) HIV-1 DNA vaccine and IL-2 plasmid, separately. All the groups were immunized 3 times at a 2-week interval. Fourteen days after the last DNA vaccine injection, recombinant MVA was injected into all the mice except those in group 1. ELISA and ELISPOT were employed to investigate the effect of IL-2 on DNA vaccine immune responses. The obtained results strongly indicate that the efficacy of HIV vaccine can be enhanced by co-administration of a plasmid encoding IL-2.

HPV-16 E6E7抗原表位嵌合体DNA疫苗黏膜免疫抑制宫颈癌的效果研究

目的 评价泛素及热休克蛋白70(HSP 70)C融合人乳头瘤病毒(HPV)16型E6E7表位嵌合体DNA疫苗鼻内黏膜免疫对宫颈癌移植瘤的预防和治疗效果。方法 建立TC-1小鼠肿瘤模型,以壳聚糖为发送载体,通过滴鼻免疫给药免疫C57BL/6小鼠。MTT法和淋巴毒性T细胞(CTL)反应检测小鼠脾脏T淋巴细胞增殖、流式细胞术检测细胞因子表达水平、肿瘤生长曲线和荷瘤小鼠存活时间评价壳聚糖包裹的HPV-16 E6E7嵌合体DNA疫苗鼻内黏膜免疫后对HPV-16型相关宫颈癌移植瘤的预防和治疗效果。结果 与对照组pcD-UH相比,实验组pcD-UE和pcD-UEH均具有显著的CTL反应,延缓HPV-16型相关宫颈癌移植瘤生长,但对已生成肿瘤无影响。鼻内黏膜途径发送壳聚糖包裹HPV-16 E6E7抗原表位嵌合体DNA疫苗仅能产生较弱的细胞免疫应答。结论 HPV-16 E6E7抗原表位嵌合体DNA疫苗通过鼻腔免疫,具有一定的肿瘤治疗和显著的肿瘤预防效果,为新型HPV疫苗的研制奠定了基础。

Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines

H5N1 鸟的流行性感冒病毒(A/chicken/Hubei/489/2004 ) 的 M 和 NP 基因被 RT-PCR 从病毒的 RNA 放大，并且分别地克隆向量进 pMD18-T。包含 M 基因(pHM6-m ) 或 NP 基因(pHM6-np ) 的表示 plasmid 然后被把 M 或 NP 基因插入到 pHM6 优核质表示向量构造；构造 plasmid 然后被定序。32 只 BALB/c 老鼠(6-week-old ) 在随机被划分成四个组。三组 BALB/c 老鼠被接种一次有 plasmid pHM6-m， plasmid pHM6-np 的 30 渭 g 或 plasmid pHM6-m (15 渭 g ) 和 pHM6-np (15 渭 g ) 的混合的任何一个 30 渭 g 的肌内的线路分别地。老鼠的一个另外的组作为控制与 100 渭 l PBS 被注射。二个星期以后，所有老鼠与相应 H5N1 鸟的流行性感冒病毒被质问，并且在下列 12 天内观察了。在 pHM6-m 组， pHM6-np 组和混合 plasmids 组的老鼠的幸存率分别地是 62.5% ， 25.0% 和 50.0% 。结果证明有效保护能被 pHM6-m 或 pHM6-np 提供，但是 pHM6-m 比 pHM6-np 提供了更好保护的效果。关键词 H5N1 流行性感冒病毒 - M 基因 - NP 基因 - 克隆 - DNA 疫苗的 CLC 数字 S852.65 基础条款：国家基本科学才能训练资助(NSFC J0630648 )