LMP2-DC Vaccine Elicits Specific EBV-LMP2 Response to Effectively Improve Immunotherapy in Patients with Nasopharyngeal Cancer / 生物医学与环境科学（英文）

Objective@#To evaluate the safety and effectiveness of a vaccine based on latent membrane protein 2 (LMP2) modified dendritic cells (DCs) that boosts specific responses of cytotoxic T lymphocytes (CTLs) to LMP2 before and after intradermal injection in patients with nasopharyngeal carcinoma (NPC).@*Methods@#DCs were derived from peripheral blood monocytes of patients with NPC. We prepared LMP2-DCs infected by recombinant adenovirus vector expressing LMP2 (rAd-LMP2). NPC patients were immunized with 2 × 10 @*Results@#We demonstrated that DCs derived from monocytes displayed typical DC morphologies; the expression of LMP2 in the LMP2-DCs vaccine was confirmed by immunocytochemical assay. Twenty-nine patients with NPC were enrolled in this clinical trial. The LMP2-DCs vaccine was well tolerated in all of the patients. Boosted responses to LMP2 peptide sub-pools were observed in 18 of the 29 patients with NPC. The follow-up data of 29 immunized patients from April, 2010 to April 2015 indicated a five-year survival rate of 94.4% in responders and 45.5% in non-responders.@*Conclusion@#In this pilot study, we demonstrated that the LMP2-DCs vaccine is safe and effective in patients with NPC. Specific CTLs responses to LMP2 play a certain role in controlling and preventing the recurrence and metastasis of NPC, which warrants further clinical testing.

Subversion of immunoproteasome subunit expression in dengue virus serotype 2-infected HepG2 cells

Abstract: INTRODUCTION: Infection with all serotypes of dengue virus (DV) results in augmented antigen presentation by MHC class I molecules. However, the upregulation of immunoproteasome subunits only results from infection with two serotypes. This study aims to elucidate changes in the expression of immunoproteasome subunits resulting from infection with DV, particularly DV serotype 2 (DV2). METHODS: HepG2 cells were grown in various culture milieu. Total cellular RNA and proteins were extracted and quantified. RESULTS: Results demonstrated sequestration of immunoproteasome subunits LMP2 and LMP7 in DV2-infected cells. CONCLUSIONS: This study provides insights into the mechanisms underlying immune evasion by DV.

Evaluation of the immunogenicity of a rBCG expressing GM-CSF and LMP2A / 中华微生物学和免疫学杂志

Objective To identify the expression of a fusion gene GCA formed from GM-CSF gene and LMP2A gene of Epstein-Barr virus (EBV) in a recombinant BCG (rBCG) and to study its immunoge-nicity.Methods The rBCG was constructed to express the fusion gene GCA and the expressed products were detected by Western blot assay .ELISA was performed to measure specific antibody titers in serum sam-ples from mice immunized with rBCG .Lactate dehydrogenase assay was used to analyze the cellular immuni-ty of mice.A mouse model of EBV-positive gastric carcinoma was established to evaluate the therapeutic effects of rBCG.Results The target proteins of GM-CSF and LMP2A were successfully expressed in rBCG . The specific antibodies were detected in rBCG immunized mice as indicated by ELISA .The maximum anti-body titer reached 1 ∶27 900 [(326.5±7.8) pg/ml] as injection with rBCG 5×108/mouse.The rBCG in-duced cytotoxicity of cytotoxic lymphocytes (CTLs) to EBV-positive gastric carcinoma cells (GT39) (with a killing rate of 89.6%±6.8%) was significantly higher than that of control group (P<0.05) The sizes of tumor in PBS control group [(1964.0±548.7) mm3] and BCG group [(1268.65±72.4) mm3] were big-ger than those in rBCG group [(168.64±78.80) mm3].Conclusion The rBCG expressing GM-CSF and LMP2A fusion gene was successfully constructed .The rBCG could induce humoral and cellular immune re-sponses in mice and inhibit the growth of tumor .

Construction and identification of recombinant retroviral vector and stable cell line expressing latent membrane protein 2A of Epstein-Barr virus / 细胞与分子免疫学杂志

AIM: To clone EBV-LMP2A gene, construct and identify the recombinant retroviral vector and stable cell strains expressing EBV LMP2A. METHODS: The full-length EBV LMP2A gene was generated by RT-PCR amplification from B95.8 cells which contain complement nucleotide sequence of EBV LMP2A gene. The gene was ligated to T-vector and sequenced to construct retroviral vector consisting with LMP2A. To produce retroviral virus, packing cells, 293T cells were co-transfected with recombinant retroviral expression vector pGEZ-LMP2A and two auxiliary viral vectors pHIT456 and pHIT60 by lipofectAMINE2000. Viral titration was performed according to the instructions of the manufacturer. To establish L929 cell line stable expressing LMP2A, L929 cells were infected with recombinant retrovirus three times and selected by Zeocine. The Zeocine-resistant clones (L929/LMP2A) were screened for LMP2A expression by RT-PCR and Western blot. RESULTS: The recombinant retrovirus vector carrying LMP2A gene was constructed successfully. Transfection yield a titer of 5×10~8 infectious particles/L. The infected L929 cells were selected by Zeocine. Results of RT-PCR and Western blot indicated that L929 transgenetic cells could stably express EBV-LMP2A. CONCLUSION: The L929 cell line stably expressing LMP2A provides suitability for extraction of the LMP2A protein and preparations of the vaccine for the therapy of EBV-associated diseases.

Generation and functional analysis of EBV LMP2A specific CTL elicited by DC transfected with recombinant adenovirus in vitro / 中国免疫学杂志

Objective:Generation and functional analysis of EBV LMP2A specific CTL elicited by DC transfected with recombinant adenovirus in vitro .Methods:PBMC were isolated from healthy EBV carriers and NPC patients, and then cocultured with autologous mature Ad5 LMP2A transfected DC at the ratios of 20∶1. Cytotoxicity of LMP2A specific CTL was determined with LDH release assay, the populations of CTL were performed by FACS,the IFN ? secretion and FasL mRNA expression of the CTL were detected by biological activity assays and RT PCR, respectively.Results:The results showed that high cytotoxicity of LMP2A specific CTL could be elicited by autologous transfected DC. The cytotoxicity boosted with the increase of transfected DC stimulation times, but there were no significant changes between two and three stimulations.The phenotypic analysis demonstrated that the LMP2A specific CTL induced at day 14 consisted of a majority of CD4 + and CD8 +T cells, and only a small percentage of CD56 + cells. The IFN ? secreted in the supernatants of cell culture also increased with the stimulation times. In addition, the specific CTL at day 14 from EBV healthy carriers could express FasL mRNA.Conclusion:Strong and functional EBV specific CTL could be generated by autologous mature DC transfected with adenovirus encoding LMP2A, which could provide a rationale for the immunotherapy of EBV associated NPC. [

Generation and expression of Epstein-Barr Virus Latent membrane protein 2A recombinant adenovirus / 中国免疫学杂志

Objective:To generate Epstein-Barr virus(EBV) Latent Membrane Protein 2A(LMP2A) recombinant adenovirus,and provide for further investigation on the therapy vaccine against EBV associated malignancies.Methods:Full length cDNA of encoding LMP2A of EBV had been amplified by reverse transcription-PCR and cloned into pGEM-T vector.The encoding cDNA of LMP2A was inserted into E1,E3-substituted adenovirus vector pAX1CW,then the LMP2A recombinant adenovirus vector was contransfected into 293 cells togetherwith EcoT221 digested Ad5-TPC.The LMP2A recombinant adenovirus was generated by homologous recombination,and primarily identificated by ClaI enzyme digestion.The expression of LMP2A on CV1 cells infected with recombinant adenovirus analyzed by fluorescence-activated cell sorting(FACS) and confocal microscope.Results:The replication-deficient LMP2A recombinant adenovirus was generated efficiently with the titers of 2.3?10 8 pfu/ml.The LMP2A could be seen on CV1 cells membrane with confocal microscope 48 h post infected with recombinant adenovirus and the percentage of CV1 cells expressing LMP2A was 94.4% by means of FACS analysis.Conclusion:These suggested that LMP2A could be expressed efficiently by recombinant adenovirus mediated transfer,and it was the foundation of further researching in its function and developing the suitable genetic engineering vaccine against EBV associated malignancies.