Recombinant Vaccinia Virus is an Effective and Non-perturbing Vector for Human Dendritic Cells Transfected with Epstein-Barr Virus Latent Membrane Protein 2A

ObjectiveTo study the effects of dendritic cells (DC) transfected with recombinant vaccinia virus encoding Epstein Barr virus (EBV) latent membrane protein 2A(LMP2A) gene,and to provide evidence for further investigation on the therapeutic vaccines against EBV associated malignancies. MethodsMature DC were transfected with EBV LMP2A recombinant vaccinia virus (rVV LMP2A). Before and after the transfection,the expression of surface antigens on mature DC including CD1a,CD83,CD40,CD80,HLA DR was measured by fluorescence activated cell sorter (FACS) and the function of DC to stimulate allogeneic T cells proliferation was measured by mixed leukocyte reactions (MLR). ResultsLMP2A protein was highly expressed (66.1 %) in DC after the transfection of rVV LMP2A. No significant changes in the primary surface antigens expression and in the MLR were detected during the transfection. Transfected DC still had strong potential in stimulating the proliferation of allogeneic T cells. ConclusionRecombinant vaccinia virus was an effective and non perturbing vector to mediate the transfection of LMP2A into DC. The functions of mature DC were not affected significantly by the transfection of Vac LMP2A. This study could provide evidence for the further immunotherapy of EBV associated malignancies,e.g. nasopharyngeal carcinoma (NPC).

Computational Prediction and Identification of Epstein-Barr Virus Latent Membrane Protein 2A Antigen-Specific CD8^+ T-Cell Epitopes

Epstein-Barr virus （EBV） associated nasopharyngeal carcinoma （NPC） is a high incidence tumor in Southeast Asia. Among EBV encoded proteins, latent membrane protein 2A （LMP2A） is an important antigen for T cell therapy of EBV. In this study, we predicted six HLA-A2 restricted CTL candidate epitopes of LMP2A by SYFPEITHI, NetMHC and MHCPred methods combined with the polynomial method. Subsequently, biological functions of these peptides were tested by experiments in vitro. In ELISPOT assay, the positive response of the LMP2A specific CTL stimulated by three （LMP2A264.272, LMP2A426-434 and LMP2A3s6.364） of six peptides respectively showed that the numbers of spots forming cells （SFC） ranged from 55.7 to 80.6 SFC/5 x 104 CO8^＋ T cells and the responding index （RI） ranged from 5.4 to 7. These three epitope-specific CTLs could effectively kill specific HLA-A2- expressing target cells. As a result, LMP2A264.272 （QLSPLLGAV）, LMP2A426.434 （CLGGLLTMV） and LMP2A356.364 （FLYALALLL） were identified as LMP2A-specific CD8^＋ T-cell epitopes. It would be useful to clarify immune response toward EBV and to develop a vaccine against EBV-correlative NPC.

Potent Dendritic Cell Vaccine Loaded with Latent Membrane Protein 2A (LMP2A)

Epstein-Barr virus （EBV）, a potential oncogenic herpesvirus, has been found to be associated with several malignancies. It＇s critical to elicit cellular immunity of the body to fight against EBV-associated tumor development. Using dendritic cells （DCs） loaded with latent membrane protein 2A （LMP2A） to elicit T cell response against tumor may be one of the most direct and safest immunotherapy approaches. The present study aimed to develop DCs-based cancer vaccine （DC loaded with LMP2A protein） and study its biological characteristics and immune functions. Purified LMP2A protein was extracted from a cell line L929/LMP2A stably expressing LMP2A. LMP2A could be loaded on DCs with no significant changes of the DC surface markers and cytomorphology. The percentage of DCs loaded with LMP2A was above 80%. LMP2A-loaded DCs markedly enhanced the proliferation of antigen-specific CD8^＋ T and CD4^＋ T cells by 3H-TdR incorporation assay. Besides, the specific cytotoxicity of the CTLs against LMP2A target cells was also significantly increased. These results indicated that DC-based vaccine loaded with virus antigen could elicit potent CTL response and provide a foundation for further study on the DC-based immunotherapy for nasopharygeal carcinoma and other EBV associated tumors. Cellular ＆ Molecular Immunology.

Chimerically fused antigen rich of overlapped epitopes from latent membrane protein 2 （LMP2） of Epstein-Barr virus as a potential vaccine and diagnostic agent

Epstein-Barr virus （EBV） is prevalent throughout the world and is associated with several malignant diseases in humans. Latent membrane protein 2 （LMP2） of EBV plays a crucial role in the pathogenesis of EBV-associated tumors; therefore, LMP2 has been considered to be a potential immunodiagnostic and immunotherapeutic target. A multi-epitope-based antigen is a promising option for therapeutic vaccines and diagnoses of such malignancies. In this study, we systematically screened cytotoxic T lymphocyte （CTL）, helper T cell （Th） and B-cell epitopes within EBV-LMP2 using bioinformatics. Based on the screen, two peptides rich in overlapping epitopes of both T cells and B cells were selected to construct a plasmid containing the sequence for a chimeric multi-epitope protein referred to as EBV-LMP2m, which is composed of LMP2aa195-232 and LMP2aa419-436. The EBV-LMP2m protein was expressed in E. coil BL21 （DE3） after prokaryotic codon optimization. Inoculation of the purified chimeric antigen in BALB/c mice induced not only high levels of specific IgG in the serum and secretory IgA in the vaginal mucus but also a specific CTL response. By using purified EBV-LMP2m as an antigen, the presence of specific IgG in the serum specimens of 202 nasopharyngeal carcinoma （NPC） patients was effectively detected with 52.84% sensitivity and 95.40% specificity, which represents an improvement over the traditional detection method based on VCA-IgA （60.53% sensitivity and 76.86% specificity）. The above results indicate that EBV-LMP2m may be used not only as a potential target antigen for EBV-associated tumors but also a diagnostic agent for NPC patients.

Construction and humoral immune response of Epstein-Barrvirus latent membrane protein 2 DNA vaccine in mice

We constructed a eukaryotic expression plas-mid encoding Epstein-Barr virus latent membrane protein 2(EBV,LMP2)and evaluated its effects on humoral immunity.First,the encoding sequence of the EBV LMP2 was amplified from B95-8 cell RNA by reverse transcrip-tion polymerase chain reaction(RT-PCR)and then was directionally cloned into eukaryotic expression vector pcDNA3.1.It was employed to evaluate immune response of the mice inoculated doubly with the DNA vaccine.The serum antibody against LMP2 was detected with enzyme-linked immunosorbent assay(ELISA).The recombinant plasmid pcDNA3.1-LMP2 was confirmed by the restrictive endonuclease analysis and sequence analysis.The serum titer of IgG antibody against LMP2 epitope in the mice immunized with the DNA vaccine encoding LMP2 was up to 1∶4000.In conclusion,the EBV LMP2 DNA vaccine can induce a strong humoral immune response in mice.

Immunotherapy of Epstein-Barr Virus Associated Malignancies Using Mycobacterial HSP70 and LMP2A356-364 Epitope Fusion Protein

Epstein-Barr virus infection is strongly associated with a number of malignancies.The EBV latent membrane protein 2A has been implicated as one of the most attractive candidates for immunotherapy of related malignancies.In previous studies,the T cell epitopes of LMP2A have been identified systematically.However,the epitope-based vaccine generally meets inefficient immunogenicity when used in vivo directly,which could be overcome by combination with appropriate adjuvants.Heat shock protein is a natural chaperon,which is able to activate the classical major histocompatibility complex class I antigen-processing pathway(cross-presentation).In this study,a minigene encoding LMP2A356-364(FLYALALLL)was genetically fused to the carboxy-terminal of mycobacterial heat shock protein 70.The epitope fusion protein was expressed and purified,and the cross-presentation of LMP2A_(356-364) by monocyte-derived dendritic cells pulsed with the epitope fusion protein was evaluated.Results showed that the epitope fusion protein-pulsed mDCs were much more efficient than the single peptide-pulsed mDCs on CTL activation.Immunization of HLA-A2.1 transgenic mice with MtHsp70-LMP2A_(356-364) generated peptide specific CTL more effectively than a single peptide plus incomplete Freund's adjuvant(IFA).Growth of LMP2A expressing B16 melanoma tumor cells was suppressed in the vaccinated groups.Our results suggested that MtHsp70-LMP2A_(356-364) fusion protein was more effective than the CD8^(+)T cell epitope alone on anti-tumor immunity.As a result,the MtHsp70-LMP2A_(356-364) fusion protein is considered to be a promising candidate vaccine for EBV related malignancies.

A lipid-based LMP2-mRNA vaccine to treat nasopharyngeal carcinoma

Nasopharyngeal carcinoma(NPC)is a serious and highly invasive epithelial malignancy that is closely associated with Epstein‒Barr virus(EBV).Due to the lack of therapeutic vaccines for NPC,we selected EBV latent membrane protein 2(LMP2)as a preferable targeting antigen to develop a lipid-based LMP2-mRNA(mLMP2)vaccine.Full-length mLMP2 expressing LMP2 was first synthesized using an in vitro transcription method and then encapsulated into(2,3-dioleacyl propyl)trimethylammonium chloride(DOTAP)-based cationic liposomes to obtain the mRNA vaccine(LPX-mLMP2).The cell assays showed that the antigenpresenting cells were capable of highly efficient uptake of LPX-mLMP2 and expression of LMP2.LMP2 could subsequently be presented to form the peptide-major histocompatibility complex(pMHC).Furthermore,LPX-mLMP2 could accumulate in the spleen,express antigens,promote the maturation of dendritic cells and stimulate antigen-specific T-cell responses in vivo.It dramatically inhibited the tumor growth of the LMP2-expressing tumor model after three doses of vaccination.Additionally,the proliferation of antigen-specific T cells in the tumor site made a good sign for the promise of mRNA vaccines in virus-induced cancer.Overall,we provided a newly developed antigen-encoding mRNA vaccine with advantages against NPC.We also demonstrated that mRNA vaccines are attractive candidates for cancer immunotherapy.

Analysis of Epstein-Barr viral DNA load, EBV-LMP2 specific cytotoxic T-lymphocytes and levels of CD4^＋CD25^＋ T ceils in patients with nasopharyngeal carcinomas positive for IgA antibody to EBV viral capsid antigen

Background Epstein-Barr virus （EBV） is a herpesvirus commonly associated with several malignant diseases including nasopharyngeal carcinoma （NPC）, which is a common cancer in Southeastern Asia. Previous studies showed that plasma levels of EBV-DNA might be a sensitive and reliable biomarker for the diagnosis, staging and evaluating of therapy for NPC. There are a few analyses of the levels of EBV-latent membrane protein 2 （LMP2）-specific cytotoxic T-lymphocytes （CTLs） in patients with NPC. This study was conducted to investigate the levels of EBV-LMP2-specific CTLs, EBV-DNA load and the level of CD4^＋CD25^＋T cells in such patients. Methods From February 2006 to April 2006, 62 patients with NPC, 40 healthy virus carriers positive for EBV viral capsid antigen （EBV-IgA-VCA） and 40 controls were enrolled in the study. We used a highly sensitive ELISPOT assay, real-time polymerase chain reaction （PCR） and flow cytometry to measure the EBV-LMP2-specific CTL response, the EBV DNA load and the level of CD4^＋CD25^＋T cells, respectively. Results The EBV-LMP2-specific CTL responses of the samples from the control, healthy virus carriers and patients with NPC were significantly different from the LMP2 epitopes, with the control and healthy virus carrier samples displaying a stronger response in three cases. There were significant differences in EBV DNA load in serum between NPC and the healthy groups; patients with NPC at stages Ⅲ or Ⅳ had significantly higher viral loads compared with those at stages Ⅰ or Ⅱ. A significantly higher percentage of CD4^＋CD25^＋ T lymphocytes were detected in the patients, compared with healthy virus carriers and healthy controls. Moreover, patients with advanced stages of NPC （Ⅲ and Ⅳ） had significantly higher percentages than the patients with early stages （Ⅰ and Ⅱ）. Conclusions Patients with NPC are frequently unable to establish or maintain sufficient immunosurveillance to control proliferating B cells harboring EBV and to destroy the tumor cells that express immunodominant LMP2 proteins. Controlling the activity of CD4^＋CD25^＋T cells and elevating CD8^＋ cells specific for LMP2 epitopes could be an effective immunotherapy for patients with NPC.