More than 99% of cervical cancers have been associated with human papillomaviruses (HPVs), particularly HPV type 16. The clear association between HPV infection and cervical cancer indicates that HPV serves as an ideal target for development of preventive and therapeutic vaccines. Although the recently licensed preventive HPV vaccine, Gardasil, has been shown to be safe and capable of generating significant protection against specific HPV types, it does not have therapeutic effect against established HPV infections and HPV-associated lesions. Two HPV oncogenic proteins, E6 and E7, are consistently co-expressed in HPV-expressing cervical cancers and are important in the induction and maintenance of cellular transformation. Therefore, immunotherapy targeting E6 and/or E7 proteins may provide an opportunity to prevent and treat HPV-associated cervical malignancies. It has been established that T cell-mediated immunity is one of the most crucial components to defend against HPV infections and HPV-associated lesions. Therefore, effective therapeutic HPV vaccines should generate strong E6/E7-specific T cell-mediated immune responses. DNA vaccines have emerged as an attractive approach for antigen-specific T cell-mediated immunotherapy to combat cancers. Intradermal administration of DNA vaccines via a gene gun represents an efficient way to deliver DNA vaccines into professional antigen-presenting cells in vivo. Professional antigen-presenting cells, such as dendritic cells, are the most effective cells for priming antigen-specific T cells. Using the gene gun delivery system, we tested several DNA vaccines that employ intracellular targeting strategies for enhancing MHC class I and class II presentation of encoded model antigen HPV-16 E7. Furthermore, we have developed a strategy to prolong the life of DCs to enhance DNA vaccine potency. More recently, we have developed a strategy to generate antigen-specific CD4+ T cell immune responses to further enhance DNA vaccine potency. The impressive pre- clinical data generated from our studies have led to several HPV DNA vaccine clinical trials.
Female ; Humans ; Oncogene Proteins, Viral/genetics/immunology ; Papillomaviridae/*genetics/immunology ; Papillomavirus Infections/immunology/*prevention & control ; Papillomavirus Vaccines/*administration & dosage ; Repressor Proteins ; Uterine Cervical Neoplasms/*prevention & control ; Vaccines, DNA/*administration & dosage ; Viral Vaccines/administration & dosage

OBJECTIVESTo study antibody response to a hepatitis B DNA vaccine by formulation with aluminum phosphate in mice.

METHODSAn eukaryotic expression plasmid inserted HBsAg gene (pcDNA3.1-S) was constructed by cloning technique and the accuracy of the construct was confirmed by restriction enzyme digestion and DNA sequencing, then hepatitis B DNA vaccine formulations were prepared by mixing pcDNA3.1-S with various concentration of aluminum phosphate in 0.9% NaCl. HBsAg expressions were assayed by ELISA in vivo five days after intramuscular injection of pcDNA3.1-S with or without aluminum phosphate. And serum samples were obtained from individual immunized or control mice 6 weeks post injection. Then anti-HBs were assayed in mice sera by ELISA.

RESULTSFive days after intramuscular immunization, the levels of HBsAg expression of groups with aluminum phosphate showed no difference from those of control group in tibialis arterials muscles. In sera, HBsAg could not be detectable in all groups. Intramuscular immunization of BABL/C mice with pcDNA3.1-S mixed aluminum phosphate (0microg, 1microg, 10microg, 50microg, 100microg) 6 weeks later, the P/N values of anti-HBs in sera were 11.54+/-5.60, 11.00+/-6.62, 20.30+/-10.20, 49.18+/-24.40 and 48.68+/-27.78, respectively. It showed that pcDNA3.1-S mixing with aluminum phosphate could increase anti-HBs titers in mice.

CONCLUSIONNo increase of HBsAg expression was observed by mixing plasmid pcDNA3.1-S with various concentration of aluminum phosphate in vivo. But Intramuscular immunization of BALB/C mice with pcDNA3.1-S mixing aluminum phosphate adjuvant can increase anti -HBs titers. It seemed that aluminum phosphate would be valuable for further investigation as a potential adjuvant of hepatitis B DNA vaccines.

Adjuvants, Immunologic ; administration & dosage ; Aluminum Compounds ; administration & dosage ; Animals ; Female ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B Vaccines ; administration & dosage ; immunology ; Mice ; Mice, Inbred BALB C ; Phosphates ; administration & dosage ; Vaccines, DNA ; administration & dosage ; immunology

Adjuvants, Immunologic ; administration & dosage ; Administration, Intranasal ; Animals ; Antibodies, Viral ; blood ; Bacterial Toxins ; administration & dosage ; Capsid Proteins ; Enterotoxins ; administration & dosage ; Escherichia coli Proteins ; administration & dosage ; Female ; Hemagglutination Inhibition Tests ; Human papillomavirus 16 ; immunology ; Immunization ; Interferon-gamma ; biosynthesis ; Lymphocyte Activation ; Mice ; Mice, Inbred C57BL ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomavirus Vaccines ; Vaccines, DNA ; administration & dosage ; immunology ; Viral Vaccines ; administration & dosage ; immunology ; Virion ; immunology

OBJECTIVETo study the effects of DNA vaccine transdermal delivery with microneedle array.

METHODSThe pcDNA3.1-HPV16E7 recombinant vector acting as gene vaccine was established. The infiltration quantity of pcDNA3.1-HPV16E7 getting across the microchannels generated by microneedle arrays in vitro was observed. 30 BALB/c mice were divided into 3 groups (experimental group, in vain plasmid group, negative control). Each group had 10 mice. Then immunized BALB/c mice with a dose of 200 microg with microneedle array every two weeks. The control groups did the same as that as the study groups. Two weeks after the third immunization, the serum and lymphocytes were separated to detect the functions of humoral immunity with indirect immunofluorescence test, while, the functions of cellular immunity with lymphocyte transformation test was also detected.

RESULTSThe DNA vaccine could easily get across the microchannels generated by microneedle arrays in vitro. Moreover, the course was permanent and the whole infiltration quantity was comparatively high, reaching 0.73819 mg/cm2 at the 30th hour. And among immunized BALB/c mouse, DNA vaccine transdermal delivery with microneedle array could induce specific antibodies. Lymphocyte transformation test showed that there was significant difference for the lymphocyte transformation rate between experiment (the average of lymphocyte transformation rate was 47.25%) and control group (the average of lymphocyte transformation rate was 30.00%) (chi2 = 12.903, P < 0.001). Also, the difference was found between in vain plasmid group (the average of lymphocyte transformation rate was 43.00%) and negative control(chi2 = 7.292, P = 0.007). While, no difference was observed in the experimental group and in vain plasmid group (chi2 = 0.817, P = 0.366).

CONCLUSIONThe DNA vaccine combined administering with microneedle array might get across the microchannels generated by microneedle arrays in vitro and induce humoral and cellular immune response in vivo.

Administration, Cutaneous ; Animals ; Human papillomavirus 16 ; genetics ; immunology ; Injections ; Mice ; Mice, Inbred BALB C ; Skin Absorption ; Vaccines, DNA ; administration & dosage ; immunology

BACKGROUNDMucosal immunity is important to defense against dental caries. To enhance mucosal immunity, a DNA vaccine mucosal delivery system was prepared by encapsulating anticaries DNA vaccine (plasmid pGJA-P/VAX) in chitosan under optimal conditions and the characteristics of the microparticles was investigated. Furthermore, the release properties and protective action of microparticles for plasmid were studied in vitro.

METHODSPlasmid loaded chitosan microparticles were prepared by complex coacervation. Three factors, concentration of DNA, sodium sulfate, and the chitosan/DNA ratios in complexes [better expressed as N/P ratio: the number of poly nitrogen (N) per DNA phosphate (P)] influencing preparation were optimized by orthogonal test. The characteristics of microparticles were evaluated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). DNA release rate of microparticles in similar gastro fluid (SGF) or similar intestinal fluid (SIF) at 37 degrees C was determined by ultraviolet spectrophotometry.

RESULTSHigh encapsulation efficiency (96.8%) was obtained with chitosan microparticles made under optimal conditions of 50 mmol/L Na2SO4, 200 microg/ml DNA and N/P ratio of 4. The size of particles was about 4 to 6 microm. The encapsulation process did not destroy the integrity of DNA. When incubated with SIL, after a release of about 10% in the first 60 minutes, no further DNA was released during the following 180 minutes. When incubated with SGL, the microparticles released a small burst (about 11%) in the first 60 minutes, and then slowly released at a constant, but different rate.

CONCLUSIONSThese chitosan microparticles showed suitable characteristics in vitro for mucosal vaccination and are therefore a promising carrier system for DNA vaccine mucosal delivery.

Animals ; CHO Cells ; Cell Survival ; Chitosan ; administration & dosage ; Cricetinae ; Drug Delivery Systems ; Electrophoresis, Agar Gel ; Immunity, Mucosal ; Particle Size ; Vaccines, DNA ; administration & dosage ; metabolism

To control coccidiosis without using prophylactic medications, a DNA vaccine targeting the gametophyte antigen Gam56 from Eimeria maxima in chickens was constructed, and the immunogenicity and protective effects were evaluated. The ORF of Gam56 gene was cloned into an eukaryotic expression vector pcDNA3.1(zeo)+. Expression of Gam56 protein in COS-7 cells transfected with recombinant plasmid pcDNA-Gam56 was confirmed by indirect immunofluorescence assay. The DNA vaccine was injected intramuscularly to yellow feathered broilers of 1-week old at 3 dosages (25, 50, and 100 microg/chick). Injection was repeated once 1 week later. One week after the second injection, birds were challenged orally with 5x10(4) sporulated oocysts of E. maxima, then weighed and killed at day 8 post challenge. Blood samples were collected and examined for specific peripheral blood lymphocyte proliferation activity and serum antibody levels. Compared with control groups, the administration of pcDNA-Gam56 vaccine markedly increased the lymphocyte proliferation activity (P<0.05) at day 7 and 14 after the first immunization. The level of lymphocyte proliferation started to decrease on day 21 after the first immunization. A similar trend was seen in specific antibody levels. Among the 3 pcDNA-Gam56 immunized groups, the median dosage group displayed the highest lymphocyte proliferation and antibody levels (P<0.05). The median dosage group had the greatest relative body weight gain (89.7%), and the greatest oocyst shedding reduction (53.7%). These results indicate that median dosage of DNA vaccine had good immunogenicity and immune protection effects, and may be used in field applications for coccidiosis control.
Animals ; Antibodies, Protozoan/blood ; Antigens, Protozoan/genetics/*immunology ; Cell Proliferation ; Chickens ; Coccidiosis/immunology/pathology/*prevention & control ; Disease Models, Animal ; Eimeria/genetics/*immunology ; Injections, Intramuscular ; Lymphocytes/immunology ; Protozoan Vaccines/administration & dosage/genetics/*immunology ; Vaccination/methods ; Vaccines, DNA/administration & dosage/genetics/*immunology

Pulse-induced permeabilization of cellular membranes, generally referred to as electroporation (EP), has been used for years as a tool to increase macromolecule uptake in tissues, including nucleic acids, for gene therapeutic applications, and this technique has been shown to result in improved immunogenicity. In this study, we assessed the utility of EP as a tool to improve the efficacy of HB-110, a novel therapeutic DNA vaccine against chronic hepatitis B, now in phase 1 of clinical study in South Korea. The potency of HB-110 in mice was shown to be improved by EP. The rapid onset of antigen expression and higher magnitude of humoral and cellular responses in electric pulse-treated mice revealed that EP may enable a substantial reduction in the dosage of DNA vaccine required to elicit a response similar in magnitude to that achievable via conventional administration. This study also showed that EP-based vaccination at 4-week-intervals elicited a cellular immune response which was about two-fold higher than the response elicited by conventional vaccination at 2-week intervals. These results may provide a rationale to reduce the clinical dose and increase the interval between the doses in the multidose vaccination schedule. Electric pulsing also elicited a more balanced immune response against four antigens expressed by HB-110: S, preS, Core, and Pol.
Animals ; Electroporation ; Hepatitis B Antigens/biosynthesis ; Hepatitis B Vaccines/administration & dosage/*immunology ; Hepatitis B, Chronic/*immunology/prevention & control ; Immunity, Cellular ; Male ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA/administration & dosage/*immunology

OBJECTIVETo evoke more effective humoral and cell-mediated immunization against hepatitis B virus (HBV) infection.

METHODSHBsAg-primed mice were boosted with HBs-DNA vaccine, and HBs-DNA-primed mice were boosted with HBsAg vaccine. Anti-HBs level was assayed by ELISA and cytotoxic T lymphocyte (CTL) response was tested by lactic acid dehydrogenase (LDH) releasing method two weeks after the boosted immunization.

RESULTSAnti-HBs level and CTL responsive rate at the effector/target cell ratio of 100:1 were 0.38 and 36% in HBsAg/HBs-DNA vaccination group, 0.32 and 27% in HBs-DNA/HBsAg vaccination group, 0.48 and 1.5% in HBsAg/HBsAg vaccination group, 0.24 and 68% in HBs-DNA/HBs-DNA vaccination group, respectively.

CONCLUSIONPriming with HBs-DNA vaccine followed by boosting with conventional HBsAg vaccine results in greater antibody response (F = 21.19, P < 0.05), and CTL response after HBsAg vaccination can be improved by boosting with HBs-DNA vaccine (F = 165.59, P < 0.05). It brings to better efficacy by combining HBsAg vaccine with HBs-DNA vaccine.

Animals ; Antibody Formation ; Hepatitis B ; immunology ; prevention & control ; Hepatitis B Antibodies ; biosynthesis ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; administration & dosage ; immunology ; Hepatitis B virus ; genetics ; Immunity, Cellular ; Mice ; Vaccines, DNA ; administration & dosage ; immunology

To investigate the adjuvant effect of granulocyte macrophage colony stimulating factor (GM-CSF) in Flaviviridae virus DNA vaccines. After DNA immunization, the antibody levels of serum from mice were detected by ELISA and indirect immunofluorescence assay. Co-immunization of GM-CSF suppressed the immune responses induced by DV1 and DV2 candidate vaccines whereas enhanced the immune response induced by HCV C and E1 DNA vaccines. As genetic adjuvant for DNA vaccines, GM-CSF might display complex diversity on the immune responses: an augmentation or suppression due to different immunogens. Therefore, GM-CSF should be used with some cautions in clinic.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Antibodies, Viral ; immunology ; DNA, Viral ; administration & dosage ; genetics ; immunology ; Dengue ; immunology ; prevention & control ; virology ; Dengue Vaccines ; administration & dosage ; genetics ; immunology ; Dengue Virus ; genetics ; immunology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; administration & dosage ; immunology ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; administration & dosage ; genetics ; immunology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

To optimize the immunization strategy against HIV-1, a DNA vaccine was combined with a recombinant vaccinia virus (rTV) vaccine and a protein vaccine. Immune responses against HIV-1 were detected in 30 female guinea pigs divided into six groups. Three groups of guinea pigs were primed with HIV-1 DNA vaccine three times, boosted with rTV at week 14, and then boosted with gp140 protein at intervals of 4, 8 or 12 weeks. Simultaneously, the other three groups of animals were primed with rTV vaccine once, and then boosted with gp140 after 4, 8 or 12 weeks. The HIV-1 specific binding antibody and neutralizing antibody, in addition to the relative affinity of these antibodies, were detected at different time points after the final administration of vaccine in each group. The DNA-rTV-gp140 immune regimen induced higher titers and affinity levels of HIV-1 gp120/gp140 antibodies and stronger V1V2-gp70 antibodies than the rTV-gp140 regimen. In the guinea pigs that underwent the DNA-rTV-gp140 regimen, the highest V1V2-gp70 antibody was induced in the 12-week-interval group. However, the avidity of antibodies was improved in the 4-week-interval group. Using the rTV-gp140 immunization strategy, guinea pigs boosted at 8 or 12 weeks after rTV priming elicited stronger humoral responses than those boosted at 4 weeks after priming. In conclusion, this study shows that the immunization strategy of HIV-1 DNA vaccine priming, followed by rTV and protein vaccine boosting, could strengthen the humoral response against HIV-1. Longer intervals were better to induce V1V2-gp70-specific antibodies, while shorter intervals were more beneficial to enhance the avidity of antibodies.
AIDS Vaccines ; administration & dosage ; genetics ; immunology ; Animals ; DNA, Viral ; administration & dosage ; genetics ; immunology ; Female ; Guinea Pigs ; HIV Infections ; immunology ; prevention & control ; virology ; HIV-1 ; genetics ; immunology ; Humans ; Immunization ; methods ; Vaccines, DNA ; administration & dosage ; genetics ; immunology ; Vaccinia virus ; genetics ; immunology ; env Gene Products, Human Immunodeficiency Virus ; administration & dosage ; genetics ; immunology