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.

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.

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.

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.

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.

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.

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.

DNA vaccines targeting amyloid-βoligomer ameliorate cognitive deficits of aged APP/PS1/tau triple-transgenic mouse models of Alzheimer’s disease

The amyloid-β(Aβ)oligomer,rather than the Aβmonomer,is considered to be the primary initiator of Alzheimer’s disease.It was hypothesized that p(Aβ3-10)10-MT,the recombinant Aβ3-10 gene vaccine of the Aβoligomer has the potential to treat Alzheimer’s disease.In this study,we intramuscularly injected the p(Aβ3-10)10-MT vaccine into the left hindlimb of APP/PS1/tau triple-transgenic mice,which are a model for Alzheimer’s disease.Our results showed that the p(Aβ3-10)10-MT vaccine effectively reduced Aβoligomer levels and plaque deposition in the cerebral cortex and hippocampus,decreased the levels tau protein variants,reduced synaptic loss,protected synaptic function,reduced neuron loss,and ameliorated memory impairment without causing any cerebral hemorrhaging.Therefore,this novel DNA vaccine,which is safe and highly effective in mouse models of Alzheimer’s disease,holds a lot of promise for the treatment of Alzheimer’s disease in humans.

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.

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.

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 )

Effects of Cytokine IL-18 Gene on Antibody Production Induced by Ag85A DNA Vaccine

Objective:To investigate the effects of plasmid containing human IL-18 gene on the humoral immune response of mice immunized by Ag85A DNA vaccines of Mycobacterium tuberculosis H 37R v strain.Methods: Human IL-18 cDNA was amplified from RNA of peripheral blood mononuclear cells(PBMCs)by RT-PCR and cloned into the pGEM-TEasy vector.After sequencing IL-18 gene was subcloned into the the sites of BamH Ⅰ and EcoR Ⅰ digestion of pcDNA3.1. BALB/c mice were injected intramuscularly with eukaryotic expression plasmid pcIL18,together with MTB pcAg85A DNA vaccines.The same immunization was repeated three times at intervals of two weeks.Mouse sera were collected at two weeks after the each injection.The titers of anti-Ag85A antibody were detected by ELISA. Results:IL-18 cDNA was amplified successfully from RNA of human PBMCs by RT-PCR and the result of sequencing was correct.The IL-18 gene was correctly inserted into the vector pcDNA3.1, which was confirmed with BamHⅠ and EcoRⅠ digestion analysis. The positive plasmid was called pcIL18.After being immunized with DNA vaccines,the titers of antibody obtained from mice being immunized by pcAg85A combining with pcIL18 were superior to mice immunized by pcAg85A independently.Conclusion:Combination of IL-18 gene with MTB pcAg85A DNA vaccine could observably enhance the humoral immune responses to pcAg85A.It remains further investigated whether IL-18 gene plus MTB pcAg85A DNA vaccine could markedly induce the cellular mediated immune response to Ag85A or not.

DNA vaccine expressing herpes simplex virus 1 glycoprotein C and D protects mice against herpes simplex keratitis

AIM： To investigate whether DNA vaccine encoding herpes simplex virus 1（HSV-1） glycoprotein C（g C） and glycoprotein D（g D） will achieve better protective effect against herpes simplex keratitis（HSK） than DNA vaccine encoding gD alone. METHODS： DNA vaccine expressing gD or gC combined g D（g D.g C） were constructed and carried by chitosan nanoparticle. The expression of fusion protein gD and gC were detected in DNA/nanoparticle transfected 293 T cells by Western-blot. For immunization, mice were inoculated with DNA/nanoparticle for 3 times with 2 wk interval, and two weeks after the final immunization, the specific immune responses and clinical degrees of primary HSK were evaluated. RESULTS： Fusion protein g D.g C could be expressed successfully in cultured 293 T cells. And, p RSC-g C.g DIL21 DNA/chitosan nanoparticle could effectively elicit strongest humoral and cellular immune response in primary HSK mice evidenced by higher levels of specific neutralizing antibody and s Ig A production, enhanced cytotoxicities of splenocytes and nature killer cells（NK）,when compared with those of gD alone or mocked vaccine immunized mice. As a result, gC-based vaccine immunized mice showed least HSK disease. CONCLUSION： gC-based DNA vaccine could effectively prevent the progress of primary HSK, suggesting that this DNA vaccine could be a promising vaccine for HSK treatment in the future.

Comparison of Five Expression Vectors for the Ha Gene in Constructing a DNA Vaccine for H6N2 Influenza Virus in Chickens

A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will be the most useful and practical for DNA vaccines in different species. This report examines the use of five expression vectors with different promoters and Kozak sequence to express the same hemagglutinin (HA) protein of an H6N2 avian influenza virus for DNA vaccination in chickens. Although intramuscular vaccination with seven DNA constructs elicited no or limited measurable H6 HA antibody responses in Hy-Line chickens, variable reduction in virus shedding for either oropharyngeal or cloacal swabs post-virus challenge were observed. This indicated that all DNA constructs generated some levels of protective immunity against homologous virus challenge. Interestingly, lower dose (50 or 100 μg) of plasmid DNAs consistently induced better immune response than higher dose (300 or 500 μg). In the transfection experiments there appeared to be a hierarchy in the in vitro expression efficiency in the order of pCAG-optiHAk/ pCAG-HAk > pCI-HAk > VR-HA > pCI-HA > pCI-neo-HA > pVAX-HA. Since the level of in vitro expression correlates with the level of immune response in vivo, in vitro expression levels of the DNA constructs can be used as an indicator for pre-selection of plasmid vaccines prior to in vivo assessment. Moreover, our results suggested that the Kozak sequence could be used as an effective tool for DNA vaccine design.

DNA Vaccines Approach:From Concepts to Applications

DNA vaccines are the third generation vaccines based on purified plasmid preparations containing transgenes that encode antigenic/therapeutic proteins or peptides capable of triggering an immune response against a wide range of diseases. This vaccine platform presents several attributes that confer distinct advantages over other vaccine technologies in terms of safety, ease of fabrication and stability. Many aspects, such as antigen expression and especially vector design, are under study because of their great influence on immunogenicity and efficacy of DNA vaccines. In this regard, with the attempt of improving the efficiency of DNA vaccines, co-expression of stimulatory sequences and diverse vector delivery systems are being optimized. With this in mind, this review aims to giving a conceptual approach of DNA vaccines, explaining their mechanisms of action and listing the already licensed veterinary DNA vaccines presented in the market.

Immunogenicity and protective efficacy of DNA vaccine against visceral leishmaniasis in BALB/c mice

The current study was designed to examine the protective efficacy of DNA vaccines based on gp63 and Hsp70 against murine visceral leishmaniasis. Inbred BALB/c mice were immunized subcutaneously twice at an interval of three weeks with pcDNA3.1 （＋） encoding T cell epitopes of gp63 and Hsp70 individually and in combination. Animals were challenged intracardially with 107 promastigotes ofLeishmania donovani 10 days post immunization and sacrificed 1,2 and 3 months post challenge. The immunized animals revealed a significant reduction （P 〈 0.05） in splenic and hepatic parasite burden as compared to the infected controls. Maximum reduction in parasite load （P 〈 0.05） was observed in animals treated with a combination ofpcDNA/gp63 and pcDNA/Hsp70. These animals also showed heightened DTH response, increased IgG2a, elevated Thl cytokines （IFN-γ and IL-2） and reduced IgG 1 and IL-10 levels. Thus, mice immunized with the cocktail vaccine exhibited significantly greater protection in comparison to those immunized with individual antigens.

Chitosan Microparticles Intended for Anti-caries DNA Vaccine Mucosal Delivery: Synthesis, Characterization and Transfection

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