OBJECTIVETo observe the regulating effect of hepatitis C virus (HCV) envelop (E) 2 gene immunization-induced immune responses by adenovirus mediated interleukin 12 (IL-12).

METHODSHCV E2 protein was expressed and purified from NIH 3T3 and then used as an antigen to detect antibodies against HCV E2. With 51Cr release, SP2/0 expressing HCV E2 was used as target cell to detect specific cytotoxic T lymphocytes (CTL) response; adenovirus recombined IL-12 was propagated by 293 cell. HCV E2 recombinant and adenovirus recombined IL-12 were injected into the quadriceps femoris muscles and abdominal cavities of 6-8 weeks old BALB/C mice. Sera were collected at 2, 3, and 4 weeks and detected for antibodies for E2. Spleen cells isolated at 4 weeks were analyzed for specific CTL response.

RESULTSIt was found that expression of IL-12 at an undetectable level did enhance HCV E2 gene immunization-induced CTL activity and there was no effect on its hormonal immune response.

CONCLUSIONUsing adenovirus to express interleukin 12 was helpful for regulation of HCV E2 gene immunization-induced immune response. Combined HCV E2 and IL-12 can render a strong anti-HCV CTL activity and may be of use in the development of HCV gene vaccine in the future.

Adenoviridae ; physiology ; Interleukin-12 ; biosynthesis ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; Viral Envelope Proteins ; genetics ; immunology

OBJECTIVETo evaluate the immune potency of recombinant adenovirus combined with rAAV1 vector expressing HPV16L1 protein in mice.

METHODSThe rAdV and rAAV1 vector containing codon-modified HPV16L1 gene was constructed using Admax and AAVmax packaging system respectively. C57 BL/6 mice were immunized with purified rAdV and rAAV1 vector through intramuscular and intranasal inoculation routes, and the titer of neutralizing antibody was determined by neutralization assay based HPV16 pseudovirus.

RESULTSIntramuscular immunization by rAAV1-mod. HPV16L1 or combined with rAd-mod. HPV16L1 can induce higher titer of neutralizing antibody in serum than that of other groups. The titer of neutralizing antibody of intranasal groups is significantly lower than that of intramuscular group, although the prime-boost strategy using in intranasal group was effective to enhance the specific humoral immunity.

CONCLUSIONThe rAAV1-mod. HPV16L1 combined with rAd-mod. HPV16L1 can induce higher titer of neutralizing antibody in serum through intramuscular route than that of other groups at the 16th week after the first immunization.

Adenoviridae ; genetics ; immunology ; Adenoviridae Infections ; immunology ; Animals ; Antibodies, Viral ; immunology ; Dependovirus ; genetics ; immunology ; Immune System Phenomena ; Immunization ; Mice ; Mice, Inbred C57BL ; Oncogene Proteins, Fusion ; immunology ; Oncogene Proteins, Viral ; immunology ; Recombinant Proteins ; genetics ; immunology

OBJECTIVETo analyze the genetic characteristics of a strain of adenovirus, Ad7d2, isolated from an infant died of severe pneumonia.

METHODSVirus isolation was performed by using the nasopharyngeal secretion from an 11-month-old infant with serious pneumonia. The viral DNA was amplified by PCR and the products were sequenced.

RESULTSOne strain of virus was isolated and was named as BJ060316-1. Sequence analysis of the hexon and fiber gene of the PCR products showed that the strain was Ad7d2, which shared 99.5% homology for 950bp hexon fragment with AF321311 Ad7d2 isolated from Israel in 1993. Blast with deduced amino acid sequence showed that BJ060316-1 lost glutamine at site 253, and at the site 495 arginine replaced serine. For fiber gene, BJ060316-1 showed 99.7% homology with AB243118 Ad7 isolated in Japan in 2005 for 975 bp fragment.

CONCLUSIONAdenovirus Ad7d2 strain BJ060316-1 isolated from a an infant with fatal pneumonia showed no virulence mutation.

Adenoviridae ; genetics ; immunology ; isolation & purification ; Adenoviridae Infections ; virology ; Fatal Outcome ; Humans ; Infant ; Male ; Pneumonia ; virology ; Viral Proteins ; genetics

The aim of this study was to evaluate the safety and efficiency of a novel, oncolytic adenovirus mutant M1 administered in conjunction with immunosuppressive agents. Animal models were established by administering purified M1 either intravenously or retroperitoneally. At different time points, blood samples were taken from the mice for testing of liver and renal function. Microscopic examination of the liver was performed to observe pathological changes. Immunohistochemical analyses were used to evaluate the expression of the adenovirus in the liver. Lymphocyte recruitment to the liver and the activation of adenovirus specific T cells were also analyzed. No signs of general toxicity were observed, but transient increases in ALT and Scr were observed following the administration of M1. Microscopic examination revealed a mild inflammatory response in the liver. Compared to intravenous injection, higher expression levels of adenoviral proteins were observed after retroperitoneal injection. Combined treatment with cyclosporine A resolved the liver and kidney dysfunction and increased the concentration of the adenovirus in the liver. The use of the novel oncolytic adenovirus mutant M1 in vivo is safe, and the combined administration of M1 with immunosuppressive agents was able to enhance the effectiveness and safety profile of M1.
Adenoviridae ; genetics ; immunology ; Animals ; Female ; Kidney ; immunology ; virology ; Liver ; immunology ; virology ; Mice ; Mice, Inbred BALB C ; Mutation ; genetics

OBJECTIVESTo define the expression of single-chain variable fragment (ScFv) against hepatitis B virus core protein (HBc) mediated by recombinant replication defective adenovirus carrying the anti-HBc ScFv gene in vitro and to define the activity of anti-HBc ScFv combining HBcAg.

METHODSThe recombinant adenoviruses carrying anti-HBc ScFv gene generated by homologous recombination in bacteria and packaged in 293 cells were transfected into HepG2 cells, and the anti-HBc ScFv was detected using SDS-PAGE and Western blot.

RESULTSGreen fluorescent protein (GFP) was observed in HepG2 cells after the transfection. SDS-PAGE displayed a protein strap about 2.7 x 10(4), and the result of Western blot displayed a positive reactive strap.

CONCLUSIONAnti-HBc ScFv can be expressed in cells mediated by recombinant replication defective adenovirus carrying the anti-HBc ScFv gene.

Adenoviridae ; genetics ; Cell Line ; Hepatitis B Antibodies ; genetics ; immunology ; Hepatitis B virus ; genetics ; immunology ; Humans ; Single-Chain Antibodies ; genetics ; immunology ; Transfection

To construct a recombinant adenovirus co-expressing the E2 protein of classical swine fever virus (CSFV) and the porcine interleukin 2 (pIL-2), the CSFV E2 gene and pIL-2 gene were amplified respectively from the plasmids pMD19-T-E2 and pMD19-T-pIL-2 by PCR. E2-pIL-2 fusion gene was obtained by using 5 consecutive glycine codons as a linker and cloned into the adenoviral shuttle plasmid AdTrack. The AdTrack-E2-pIL-2 was linearized and transformed into E. coli BJ5183 with the backbone plasmid AdEasy1. The resultant recombinant plasmid AdEasy-E2-pIL-2 was transfected into the 293 cells where the recombinant adenovirus rAd-E2-pIL-2 was produced. The immunogenicity of rAd-E2-pIL-2 was evaluated in rabbits. The results of RT-PCR and Western-blotting showed that rAd-E2-pIL-2 could carry and express E2 and pIL-2 proteins. The titer of the rAd-E2-pIL-2 was 10(8.12) PFU/mL. After immunized with rAd-E2pIL-2, The injected rabbits developed a high level of CSFV specific antibodies. Regular fever was not detected in the rAd-E2-pIL-2-immunized rabbits upon challenge with CSFV C stain, and specific lymphoproliferative responses to the CSFV was detected in the lymphocytes from the immunized rabbits. In conclusion, rAd-E2-pIL-2 was constructed successfully and it could be an attractive vaccine candidate against CSFV.
Adenoviridae ; genetics ; metabolism ; Animals ; Cell Line ; Classical swine fever virus ; genetics ; Humans ; Interleukin-2 ; genetics ; immunology ; Rabbits ; Swine ; Viral Proteins ; genetics ; immunology ; metabolism ; Viral Vaccines ; immunology

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

BACKGROUNDTo observe the LMP2 specific cellular and humoral immune responses after immunization with recombinant adenovirus Ad-LMP2 in rhesus.

METHODSThe rhesuses were immunized with Ad-LMP2 through intra muscular injection in three groups, high dosage (4.5 x 10(11) VP/kg), medium dosage (1.5 x 10(11) VP/kg) and low dosage (0.5 x 10(11) VP/kg) groups. They were totally immunized six times at intervals of 5 days. The specific cellular immune responses were tested during the 7th week by ELISPOT after immunization. And the titers of anti-LMP2 antibody were tested by EIA throughout the period of immunization.

RESULTSLMP2 induced specific cellular and humoral immune responses in all three dosage group. The potency of immune responses was related with the dosage of immunization. Higher dosage elicited more potent immune response. Both the neutralizing antibody to adenovirus and anti-LMP2 antibody could be detected from 2 weeks after immunization. They would reach the peak during 3-4 weeks after immunization, then declined during the 7th week after immunization.

CONCLUSIONThe recombinant adenovirus LMP2 could induce specific cellular and humoral responses in rhesus after immunization.

Adenoviridae ; genetics ; Animals ; Antibodies, Viral ; blood ; Antibody Formation ; immunology ; Female ; Immunization ; methods ; Macaca mulatta ; Male ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, DNA ; genetics ; immunology ; Viral Matrix Proteins ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology

OBJECTIVETo construct DNA and recombinant adenovirus vector vaccines containing an env gene from the prevalent subtype B strain in China and try to use them for therapeutic and prophylactic vaccines.

METHODSThe candidate plasmid DNA vaccine pVR-gp160 and recombinant adenovirus vaccine rAdV-gp160 were constructed separately. BALB/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gp120-specific cellular responses and antibody levels were detected by ELISPOT and ELISA respectively.

RESULTSDNA vaccine alone and combined vaccines in a DNA prime/rAdV-gp160 boost vaccination regimen induced high level of Gp120-specific cellular responses. While low level of Gp120-specific antibodies were elicited in all groups.

CONCLUSIONDNA and rAdV vaccines could efficiently express Gp160 protein and activate specific cellular responses.

AIDS Vaccines ; genetics ; immunology ; Adenoviridae ; genetics ; immunology ; Animals ; China ; Genes, env ; immunology ; Genetic Vectors ; genetics ; immunology ; HIV Antibodies ; genetics ; immunology ; HIV Envelope Protein gp120 ; genetics ; immunology ; HIV Envelope Protein gp160 ; genetics ; immunology ; HIV-1 ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; immunology ; Vaccines, DNA ; genetics ; immunology ; Vaccines, Synthetic ; genetics ; immunology

OBJECTIVETo test the infeciton efficiency of recombinant adenoviral vector carrying HBsAg-HSP70 chimeric gene and to abserve its biological characteristics.

METHODSPeripheral blood mononuclear cells (PBMC) were separated from healthy blood donor and they were infected by Ad-HSP70-HBsAg on the first day of isolation. DCs were induced in medium with cytokines IL-4, GM-CSF and TNF-alpha in vitro. The biological characteristics of DC induced were analyzed by inverted fluorecent microscope, RT-PCR, flow cytometer (FACS), and mixed lymphocyte reaction (MLR).

RESULTSThe traced gene-GFP were abserved in DCs by inverted fluorecent microscope and HSP70-HBsAg gene mRNA expression was detected by RT-PCR after the Ad-HSP70-HBsAg infection. FACS analysis shown that the expression of CD1a, CD80, CD86 and HLA-DR on surfece of two groups of DCs were similar. MLR showed that there are not a statitic difference of stimulated index (SI) between two groups.

CONCLUSIONResults indicated that Ad-HSP70-HBsAg can effectively infected DCs without affecting its biological characteristics.

Adenoviridae ; genetics ; physiology ; Cells, Cultured ; Cytokines ; genetics ; immunology ; Dendritic Cells ; immunology ; virology ; Genetic Vectors ; genetics ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Hepatitis B Surface Antigens ; genetics ; immunology ; Humans ; Lymphocyte Culture Test, Mixed ; Recombinant Fusion Proteins ; genetics ; immunology