Adenoviridae ; genetics ; Animals ; Dengue Vaccines ; immunology ; Humans ; Plasmids ; Recombinant Fusion Proteins ; immunology ; Vaccines, Attenuated ; immunology ; Vaccines, DNA ; immunology ; Vaccines, Synthetic ; immunology

OBJECTIVETo construct a humulus pollen allergy DNA vaccine pcDNA3.1-Hum and investigate its effect for immune protection mediated by Foxp3(+)Treg cells in asthmatic mice.

METHODSThe target humulus gene obtained from pTripIEx2-Hum plasmid by double enzyme digestion was inserted sequentially into pcDNA3.1(-) vector to generate the recombinant plasmid pcDNA3.1-Hum, which was validated by sequencing. The pcDNA3.1-Hum plasmid was transfected into COS-7 cells and the expression of the ectopic protein was analyzed using Western blotting. Co-cultured dendritic cells and CD4(+)CD25(-) T cells were stimulated with the expressed protein to test its efficacy in inducing Foxp3(+)Treg cells. The levels of humulus-specific IgE and IgG2a were assayed to evaluate the allergenicity and immunogenicity of pcDNA3.1-Hum in mice. The immunoprotective effect of pcDNA3.1-Hum was assessed in a mouse model of humulus-specific asthma.

RESULTSThe constructed pcDNA3.1-Hum plasmid was validated by sequencing and Western blotting, and the expressed protein was shown to induce Foxp3(+)Treg cells in the co-culture. In normal mice, pcDNA3.1-Hum induced a significant increase of humulus-specific IgG2a but had no effect on IgE. In the asthmatic mice, pcDNA3.1-Hum significantly decreased inflammatory cell counts and eosinophil percentages in the BALF, ameliorated lung inflammation, and lowered AHR and IL-4 levels; immunization of the mice with pcDNA3.1-Hum reversed humulus-induced reduction of serum IFN-γ and prevented the humulus-triggered reduction of Foxp3(+)Treg cell percentage in the spleen.

CONCLUSIONWe have successfully constructed a highly immunogenic pcDNA3.1-Hum DNA vaccine that can mediate immune protection by inducing Foxp3(+)Treg cells.

Allergens ; immunology ; Animals ; Asthma ; immunology ; Cell Differentiation ; Disease Models, Animal ; Female ; Humulus ; immunology ; Mice ; Mice, Inbred BALB C ; Plasmids ; T-Lymphocytes, Regulatory ; cytology ; immunology ; Vaccines, DNA ; immunology

OBJECTIVETo clone PIB gene of Neisseria gonorrhoeae, and to construct a recombinant eukaryotic expression vector pCI-PIB and to understand the effects of pCI-PIB vaccination in mice to induce specific humoral and cellular immune responses.

METHODSThe entire PIB gene of Neisseria gonorrhoeae (960 bp) was amplified by using PCR. An eukaryotic eukaryotic vector pCI-PIB was then constructed. BALB/c mice (n = 65, 100 microg/time/mouse) were immunized with pCI-PIB by intramuscular injection. ABC assay was employed to examine the PIB expression in muscular cells of the pCI-PIB-immunized mice (n = 10). ELISA and MTT assays were used to measure the effects of humoral and cellular immune responses of the remaining pCI-PIB-immunized mice. By using slide agglutination test and complement bacteriolytic test, the serum anti-bacterial activity of the pCI-PIB immunized mice was determined.

RESULTSThe entire PIB gene amplification fragment of the expected size (960 bp) was successfully obtained by PCR. In comparison with the reported PIB gene sequence (GenBank No: AF090801), the homology of nucleotide sequence of the target inserted fragment in the recombinant plasmid pCI-PIB was as high as 99.28%. The muscular cells of the immunized mice could take in pCI-PIB and then express PIB. In the pCI-PIB immunized mice, the higher titer (1:4000) of specific serum IgG and the specific T lymphocyte response were found. The proliferation index (4.031) was significantly higher than that of the controls (1.127) (t = 71.71, P < 0.05). The sera and washings from the pCI-PIB immunized mice could agglutinate Neisseria gonorrhoeae and kill this microbe in presence of complements.

CONCLUSIONIn this study we successfully constructed a recombinant eukaryotic expression vector pCI-PIB. The mice inoculated with pCI-PIB might efficiently produce the specific humoral and cellular immune responses, suggesting that pCI-PIB should be potential service as a candidate of Neisseria gonorrhoeae DNA vaccines.

Animals ; Antibody Formation ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; DNA, Recombinant ; immunology ; Female ; Immunity, Cellular ; Mice ; Mice, Inbred BALB C ; Neisseria gonorrhoeae ; genetics ; immunology ; Plasmids ; Vaccines, DNA ; immunology

OBJECTIVETo explore the involvement of intron A into eukaryotic expression vector to improve antigen expression efficiency and enhance immunogenicity of DNA vaccine in mice.

METHODSAs model antigen, the coding gene of mycobacterial Hsp65 was cloned into eukaryotic expression vector pCMV4.0 with intron A involved and pVAX1 without intron A involved, respectively. The resulted recombinant expression vectors were transfected into 293T cells and were then injected into BALB/c mice as DNA vaccines. Anti-Hsp65 specific IgG and isotype were detected by ELISA and T cell immune response was analyzed by enzyme-linked immunosorbent spot (ELISPOT) assay and intracellular cytokine staining.

RESULTSCompared with non-intron A pVAX1hsp65, the recombinant plasmid pCMV4.0hsp65 involved with intron A pVAX1hsp65 caused higher expression level of Hsp65 in 293T cells, and enhanced Th1 type immune response, which was defined as higher level of anti-Hsp65 specific total IgG level (3.76 ± 0.23 vs 3.15 ± 0.22, P < 0.01) and IgG2a/IgG1 ratio (4.08 ± 0.04 vs 2.23 ± 0.12, P < 0.01) and more IFN-γ-secreting CD4(+) ((2.0 ± 0.058)% vs (1.5 ± 0.087)%, t = 4.804, P < 0.01) and CD8(+) ((0.6 ± 0.058)% vs (1.0 ± 0.115)%, t = 3.098, P < 0.05) T lymphocytes. The difference showed statistical significance.

CONCLUSIONIntron A can improve the expression efficiency of mycobacterial Hsp65 antigen and enhance immunogenicity of DNA vaccine in mice when involved into eukaryotic expression vector.

Animals ; Bacterial Proteins ; genetics ; immunology ; Chaperonin 60 ; genetics ; immunology ; Female ; Genetic Vectors ; Introns ; immunology ; Mice ; Mice, Inbred BALB C ; Plasmids ; Vaccines, DNA ; genetics ; immunology

OBJECTIVETo observe the expression of a targeted fusion anticaries DNA vaccine pGJA-P in situ. To compare the levels of specific antibodies and anticaries efficacy generated by pGJA-P and pGLUA-P, a fusion anticaries DNA vaccine.

METHODSpGJA-P was administrated intramuscularly or intranasally to rats, and the expression of recombinant protein was detected by immunohistochemistry technique. Wistar rats were fed a cariogenic diet and orally infected with S. mutans, then immunized with pGJA-P or pGLUA-P via the intramuscular or intranasal route. All rats received a booster immunization 2 weeks later. At the termination of the experiment, blood and saliva samples were collected for assay of antibodies by ELISA and jaws were obtained for caries evaluation by the Keyes method.

RESULTSRecombinant protein could be detected in muscle in intramuscularly immunized rats and in nasal mucosa in intranasally immunized rats. Rats immunized intramuscularly with pGJA-P had significantly higher serum IgG levels than others (P < 0.01). Rats immunized intranasally or intramuscularly with pGJA-P had significantly higher salivary IgA levels than others (P < 0.01). Keyes scores of pGJA-P groups were significantly lower than those of pGLUA-P groups and pCI groups (P < 0.01).

CONCLUSIONSpGJA-P could be correctly expressed in vivo. pGJA-P generated increased humoral immune response and anticaries efficacy compared with pGLUA-P.

Animals ; Bacteriocin Plasmids ; immunology ; Dental Caries ; prevention & control ; Female ; Rats ; Rats, Wistar ; Recombinant Fusion Proteins ; immunology ; Streptococcus mutans ; immunology ; Vaccines, DNA ; immunology

BACKGROUNDTo determine the antigenicity of HGV NS5 recombinant proteins expressed in E.coli.

METHODSHGV NS5a,NS5b and core/NS5b fusion genes were cloned into pThioC vector. Three expression plasmids were transformed into JM109(DE3) competent cells then expressed with induction by IPTG. Western blot and ELISA were used to determine the antigenicity after the three recombinant proteins were purified.

RESULTSAfter identification by restriction enzyme and sequencing, it was confirmed that the expressed was target proteins espected. Purified expression proteins were found strongly immunoreactive among anti HGV positive sera by Western blot and ELISA. Compared with mixed recombinant antigen (including core, NS5a synthetic peptide and NS3 recombinant proteins), in the 22 positive sera detected with mixed antigen, 68%(15/22), 90%(20/22) and 73%(16/22) were positive by P5a,P5b and Pc?5b antigens; In the 70 negative samples with mixed antigen, 7%(5/70), 1%(1/70) and 6%(4/70) were positive by P5a, P5b and Pc?5b antigens. The positive alone was found among RTPCR positive specimen using these recombinant antigens.

CONCLUSIONSNS5 gene expressed in E.coli?which couldn't be covered with other regions of antigens was one of the essential epitopes to HGV immunologic diagnosis.

Antibodies, Viral ; blood ; Antigens, Viral ; blood ; Epitopes ; immunology ; GB virus C ; genetics ; immunology ; Humans ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

OBJECTIVETo construct a novel immunogene therapeutic plasmid that expresses human interleukin-12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF) and B7.1 and observe its expression in vivo and in vitro.

METHODSHuman IL-12 gene fragment was cloned into the upper stream of IRES gene in the previously constructed plasmid pVAX-IRES-GM-CSF-B7.1, and the positive recombinant plasmid pVAX-IL-12-GB was transfected into 293T cells via Lipofectamine 2000. The expressions of IL-12 and GM-CSF-B7.1 mRNA and proteins in the transfected cells were assayed by RT-PCR and ELISA, and B7.1 expression was tested by fluorescence-activated cell sorting and immunofluorescence assay. The plasmid pVAX-IL-12-GB was delivered into mouse muscle by electroporation, and the expression of IL-12 in the muscle tissue was identified by immunohistochemistry.

RESULTSEnzyme digestion, PCR and sequence analysis all confirmed successful construction of the recombinant plasmid pVAX-IL-12-GB. IL-12, GM-CSF and B7.1 expressions were all detected in transfected 293T cells, and the expression of IL-12 was also detected in the transfected mouse muscular tissues.

CONCLUSIONA novel anti-tumor immunogene vaccine constructed can be expressed both in vivo and in vitro, which facilitates further studies of tumor immunogene therapy.

Animals ; B7-1 Antigen ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; Electroporation ; Genetic Therapy ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; Humans ; Interleukin-12 ; genetics ; immunology ; Mice ; Plasmids ; Transfection

Mice and 3-day-old chickens were orally inoculated with the recombinant attenuated Salmonella typhimurium strain ZJ111 carrying pcDNA3-F expression plasmid encoding the fusion protein of Newcastle disease virus (NDV). The results showed that ZJ111/pcDNA3-F was relatively safe. The recombinant plasmid pcDNA3-F was stable within the host stain ZJ111 in vitro and in vivo as shown by restriction enzyme analysis and PCR identification of the F gene. In an experimental vaccination study, 3-day-old chickens were orally immunized with ZJ111/pcDNA3-F with a dose of 108 cfu per chicken and boosted two weeks later. At week 4 post boosting, all chickens were challenged with a lethal dose of a virulent NDV strain F48 E9. The results showed that oral vaccination with ZJ111/pcDNA3-F induced stronger humoral and cellular immune responses than intramuscular immunization with naked pcDNA3-F plasmid. It also exhibited higher protection rate than the latter (66.7% vs 50%). This study indicates that the DNA vaccine using attenuated Salmonella typhimurium as delivery carrier had good safety, stability and immunogenicity and exhibited good potential of low cost and convenience for poultry disease control.
Animals ; Chickens ; Immunity, Cellular ; immunology ; Immunity, Humoral ; immunology ; Mice ; Newcastle Disease ; immunology ; virology ; Plasmids ; Polymerase Chain Reaction ; Salmonella typhimurium ; genetics ; metabolism ; Vaccines, DNA ; adverse effects ; genetics ; immunology

Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for development of protective HCV vaccines. However, this protein may attenuate the induction of systemic immune responses due to its immunomodulatory properties. In this study, we constructed a HCV core gene-containing eukaryotic expression plamid pCI-C, and an in vivo-inducible prokaryotic expression plasmid pZW-C, and transformed the recombinant plasmids into an attenuated Salmonella typhimurium aroA strain SL7207. The resulting bacterial strains SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and the immune responses specific to HCV core protein were assessed. Immunization with the recombinant bacteria SL7207/pCI-C led to a persistent drop in percentage of CD3 CD4 T cells, and induced a weak anti-core IgG production. Splenocytes from SL7207/pCI-C immunized mice developed a relatively weak proliferation response and inferior cytotoxic activity compared to those from the mice immunized with bacteria SL7207/pZW-C. Boost immunization with SL7207/ pCI-C yielded limited improvement in immune strength, while the boost with bacteria SL7207/pZW-C significantly enhanced the immune response. These results suggest that de novo synthesis of native HCV core protein may blunt the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.
Animals ; Hepacivirus ; genetics ; immunology ; Mice ; Plasmids ; genetics ; Salmonella typhimurium ; genetics ; metabolism ; Vaccines, DNA ; immunology ; Viral Core Proteins ; genetics ; immunology ; Viral Hepatitis Vaccines ; genetics ; immunology

The fusion protein (F) gene of Newcastle disease virus was amplified by polymerase chain reaction (PCR) from the recombinant plasmid pVAX1-F, and subcloned into eukaryotic expression vector pmcDNA3. 1+. The F gene was identified by sequencing. The recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was designated as SL7207 (pmcDNA3. 1-F). In vitro and in vivo experiments showed that the plasmid stability of pmcDNA3. 1-F was apparently higher than that of pcDNA3. 1-F in SL7207. In order to compare the immune response induced by these two re combinant bacteria, BALB/c mice were immunized orally with them at the dosage of 2 x 10(9) CFU respectively. Both SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) initiated F-specific serum and mucosal antibodies in immunized mice. Furthermore, 4-day-old SPF chickens were immunized with SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) at the dosage of 5 x 10(9) CFU and boosted two weeks later with the same dosage. Humoral and intestinal mucosal immune responses were observed and their levels were significantly higher than that of negative and positive controls. The result of protective efficacy showed that the chickens immunized with SL7207(pmcDNA3. 1-F) had the protective rate of 70.0%, higher than that of the SL7207 (pcDNA3. 1-F) with 50.0%. In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium has good immunogenicity. A novel mucosal DNA vaccine has been developed and could be useful for controlling the infection and epidemic of Newcastle disease in the poultry.
Animals ; Chickens ; Female ; Immunization ; Mice ; Mice, Inbred BALB C ; Newcastle disease virus ; immunology ; Plasmids ; Salmonella typhimurium ; genetics ; Vaccines, Attenuated ; immunology ; Vaccines, DNA ; immunology ; Viral Vaccines ; immunology