基因免疫接种技术与基因免疫苗

基因免疫接种和基因免疫苗是近几年来迅速发展的研究领域，在病毒性疾病和肿瘤防治中有着广阔的应用前景。本文阐述基因免疫接种诱发宿主免疫应答的机理，概述基因免疫接种方法及做为疫苗的可行性。

Comparison of Immune Responses against FMD by a DNA Vaccine Encoding the FMDV/O/IRN/2007 VP1 Gene and the Conventional Inactivated Vaccine in an Animal Model

Foot-and-mouth disease virus （FMDV） is highly contagious and responsible for huge outbreaks among cloven hoofed animals. The aim of the present study is to evaluate a plasmid DNA immunization system that expresses the FMDV/OflRN/2007 VP1 gene and compare it with the conventional inactivated vaccine in an animal model. The VP1 gene was sub-cloned into the unique Kpn I and BamH I cloning sites of the peDNA3.1＋ and pEGFP-N1 vectors to construct the VPI gene cassettes. The transfected BHKT7 cells with sub-cloned pEGFP-N1-VP1 vector expressed GFP-VP1 fusion protein and displayed more green fluorescence spots than the transfected BHKT7 cells with pEGFP-N1 vector, which solely expressed the GFP protein. Six mice groups were respectively immunized by the sub-cloned pcDNA3.1＋-VP1 gene cassette as the DNA vaccine, DNA vaccine and PCMV-SPORT-GMCSF vector （as molecular adjuvant） together, conventional vaccine, PBS （as negative control）, pcDNA3.1＋ vector （as control group） and PCMV-SPORT vector that contained the GMCSF gene （as control group）. Significant neutralizing antibody responses were induced in the mice which were immunized using plasmid vectors expressing the VP1 and GMCSF genes together, the DNA vaccine alone and the conventional inactivated vaccine （P〈0.05）. Co-administration of DNA vaccine and GMCSF gene improved neutralizing antibody response in comparison with administration of the DNA vaccine alone, but this response was the most for the conventional vaccine group. However, induction of humeral immunity response in the conventional vaccine group was more protective than for the DNA vaccine, but T-cell proliferation and IFN-？ concentration were the most in DNA vaccine with the GMCSF gene. Therefore the group that was vaccinated by DNA vaccine with the GMCSF gene, showed protective neutralizing antibody response and the most Thl cellular immunity.

PROTECTION AGAINST LEPTOSPIROSIS BY IMMU NIZATION WITH PLASMID DNA ENCODING 33 kDa ENDOFLAGELLIN OF L.INTERROGANS SEROVAR LAI

To evaluate how the efficacy of DNA inocutation affects the ability to raise protective immunity against Leptospira. [WT5”BX]Methods.[WT5”BZ] A pair of oligonucleotide primers were designed to amplify the endoflagellar gene of L. interrogans sensu stricto serovar lai. An approximately 840bp fragment was generated with PCR and inserted into VR1012, a plasmid DNA expression vector, after the fragment and VR1012 were digested respectively with EcoRV and Sal I. A recombinant plasmid designated as VR1012+flaB2 was obtained. The vector, VR1012 consits of a pUC18 backbone with the cytomegalovirus(CMV) IE1 enhancer, promoter, and intron A, transcription regulatory elements and the BGH polyadenylation sequences driving the expressing of leptospiral endoflagellar gene of L. interrogans sensu stricto serovar lai. Plasmid encoding leptospiral endoflagellin gene was injected into quadriceps of NZW rabbits. [WT5”BX]Results.[WT5”BZ]This resulted in the generation of specific leptospiral antibody with high ELISA titer (1:32768) in the rabbits. Immuno/protection was performed in guinea pigs without adjuvant. The group“VR1012+flaB2” showed higher survival rate(90%,9/10 animals),compared with the group “VR1012 lack flaB2” and the group “normal saline”. [WT5”BX]Conclusion.[WT5”BZ]The technique of DNA vaccine has potential advantages over certain other vaccine preparation technologies. However whether DNA vaccine will be useful for vaccine development remains to be tested.

Enhanced anti-tumor immunity ex vivo induced by GM-CSF gene transducted dendritic cell vaccine

Objective： The aim of the study was to investigate whether dendritic cell （DC） precursors, recruited by injection of chemokine ligand 3 （CCL3）, induce enhanced anti-tumor immunity after granulocyte-macrophage colony stimulating factor （GM-CSF） transfection in mice ex vivo. Methods： The 615 mice were injected with CCL3 via the tail vein. Freshly isolated B220–CD11c＋ cells were cultured with cytokines. For adenoviral （Ad）-mediated gene transduction, DCs were transferred AdGM-CSF gene at different ratios of multiplicity of infection （MOI） to determine the optimal gene transfection conditions, and detecting the expression of GM-CSF after transfection. The variation of GM-CSF gene-modified DCs were analyzed by morphological observation, phenotype analysis, and mixed lymphocyte reaction （MLR）. DCs were loaded with gastric cancer antigen obtained by frozen and thawed method. The stimulated DCs vaccination induced T lymphocytes, and the killing effect of T cells to gastric cancer cells was assayed by MTT. INF-γ production was determined with the INF-γ ELISA kit. Results： B220–CD11c＋ cells numbers increased after CCL3 injection. ELISA results showed that after GM-CSF gene modification, DC could produce high level of GM-CSF. When DCs were transferred AdGM-CSF gene at MOI equal to 1：100, GM-CSF level in culture supernatants reached saturation [（130.00 ± 12.61） pg/mL]. After GM-CSF gene-modification, DCs tended to more maturated through morphological observation and were phenotypically identical to typical DC and gained the capacity to stimulate allogeneic T cells. T lymphocytes stimulated with DC transduced with GM-CSF gene showed the specific killing effect on gastric carcinoma cells and produced high level of INF-γ [（1245.00 ± 13.75） pg/mL]. Conclusion： CCL3-recruited DCs modified by adenovirus-transducted GM-CSF could produce high level of GM-CSF, which tended to more maturated, and the capacity of activating allogeneic T lymphocytes proliferation was enhanced greatly. Moreover, they could stimulate specific cytotoxic T lymphocyte （CTL） to gastric cancer ex vivo.

Anti-Tumor Efficacy of Gene Vaccine Expressing PSMA

OBJECTIVE To observe anti-tumor effects of PVAX-PSMA gene vaccine. METHODS The PSMA gene was inserted into a mammalian expression vector, PVAX-1, to construct the DNA vaccine candidate, and was then used to vaccinate C57BL/6 mice. Animals vaccinated with PVAX-1 and NaC1 were used as controls. Anti-PSMA antibody was detected in sera of the animals. The proliferation and cytotoxicity of the spleen cells were observed. The immunized mice were inoculated with RM-1 cells. The mice were inoculated with RM-1 cells, and then the mice were immunized. The anti-tumor efficacy of the gene vaccine was evaluated by the ratio of tumor formation, tumor volume, tumor mass before and after gene vaccination and evaluated by survival rate of the immunized mice. RESULTS High level of anti-PSMA antibody was induced in the PVAX-PSMA group. The splenocytes from PVAX-PSMA group were stimulated to produce strong proliferation responses and significant cytotoxic T-cells （CTL） activity. After the mice were immunized with PVAX-PSMA gene, tumor occurrence was decreased, and the growth velocity of tumor was markedly reduced, resulting in prolonged tumor-free time （P 〈 0.05）. CONCLUSION PVAX-PSMA gene vaccine has significant anti- tumor effects and provides an experimental basis for primary prevention and immunotherapy of prostate cancer.

Anti-tumor Immunity of Gene Vaccine with Nucleofection Technology

OBJECTIVE To observe enhancement of anti-tumor immunity by gene vaccine using nucleofection technology METHODS The technique of nucleofection was used to transfer effectively plasmid DNA into immature dendritic cells （iDCs）; we studied immune responses regulated by DNA vaccine using real-time quantitative polymerase chain reaction （PCR） and western-blotting to optimize the follow-up lymphocyte activation. The anti-tumor capacity of lymphocytes primed by DCs was analyzed using lactate dehydrogenase with a non-radioactive cytotoxicity assay.

Antigen Gene Cloning and Expression of HIV-1 Toward an AIDS Vaccine Design Ⅰ.Amplification and Sequencing of HIV-1 Antigen Genes

Objective:To amplify antigen genes from patients with human immunodeficiency virus type 1 (HIV-1) in Guangdong Province for candidate AIDS vaccine design. Methods:Viral nucleic acid was isolated from 10 HIV-1 infected individuals' peripheral blood collected during 1995-2000 in Guangdong Province. The viral gag p24 gene and env gp120 gene were amplified by nested-PCR and sequenced. The homologies among HIV-1 isolates were compared with HIV-BLAST. Results: Among 10 HIV-1 isolates, nine are homologous to viruses of subtype B, and one is homologous to viruses of subtype E. Conclusion: Subtype B viruses of HIV-1 are predominantly present in Guangdong Province.

Antigen Gene Cloning and Expression of HIV-1 Toward AIDS Vaccine Design Ⅱ.Subtype Classification and Quasi-species Identification of HIV-1

Objective: To analyze subtypes and quasi-species of isolatedviruses from HIV-1 infected individuals among the populationof Guangdong Province, for understanding the molecularepidemiological dynamics of local HIV-1 isolates, thus laying afoundation for designing a candidate AIDS vaccine. Methods: By hetero-duplex mobility assay (HMA) andsingle strand conformation poly-morphism (SSCP) analysison amplicons from single-primed polymerase chain reaction(SP-PCR), subtypes and quasi-species of tested HIV-1 isolateswere elucidated, and amplicons were sequenced forconfirmation. Results: Specific amplicons from different subtypes andquasi-species of HIV-1 could be discernible by HMA andSSCP analysis. Conclusion: HIV-1 isolates from different patients might beeither a different subtype or an identical subtype, and HIV-1isolates from an individual were present in a population ofquasi-species.

Immunogenicity of the recombinant adenovirus type 5 vector with type 35 fiber containing HIV-1 gag gene

The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene （rAd5/F35-mod. gag） was investigated in BALB/c mice, in which the rAd5/F35-mod, gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod, gag, rAd5-mod, gag or DNA and were boosted after 3 weeks. To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5- GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte （CTL） response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35- rood. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIV- specific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod, gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.