The initial results of Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP-1) for screening nasopharyngeal carcinoma (NPC)

Objective: Early diagnosis of nasopharyngeal carcinoma (NPC) is an important method to improve the survival rate.However,the sensitivity and specificity of the screening protocols which was widely used in clinic now are considered to be unsatisfactory.Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP-1) is one of the proteins that have been suggested to be a classic oncogene with transformation properties.The current study set out to discuss the clinical significance of LMP-1 on the screening of NPC.Methods: Three hundred patients who visited our institution (Department of Radiation Oncology,Fujian Provincial Cancer Hospital,Fuzhou,China) with ENT symptoms between 2007 and 2008 were involved in this study,and all of them were agreed to be involved in this investigation.Not only did they undergo nasopharyngeal swab to obtain cells for the LMP-1 polymerase chain reaction (PCR) analysis,but also nasopharyngeal biopsy were taken to identify the diagnosis.Results: An amount of DNA that was sufficient for PCR was extracted from 243 (81%) swab samples,the positive rate of LMP-1 of those with non-nasopharyngeal carcinoma was 3.85% (4/108),which was much lower than those with nasopharyngeal carcinoma (P < 0.05).By detecting LMP-1 in nasopharyngeal swabs,NPC was diagnosed with a sensitivity of 88.15% (119 of 135 patients),specificity of 96.30% (104 of 108 patients),a positive predictive value of 95.2% (119 of 123 patients),a negative predictive value of 86.67% (104 of 120 patients),accuracy of 91.77%,and Youden index of 84.45%.Conclusion: The nasopharyngeal swab coupled with PCR-based EBV LMP-1 detection have high sensitivity and specificity,and also good repeatability,it could serve as part of the screening program for high-risk populations.

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.

IgA／EBV—VCA阳性人群和鼻咽癌患者的EB病毒潜伏膜蛋白2特异性细胞免疫及EBVDNA载量检测

目的探讨IgA／VCA抗体滴度与EBVLMP2特异性细胞免疫水平和EBVDNA拷贝数关系。方法免疫酶法检测普查样本IgA／VCA抗体滴度；选取IgA／VCA抗体阳性人群和治疗前鼻咽癌患者，通过γ-干扰素Elispot和EBVDNA荧光定量试剂盒检测LMP2特异性细胞免疫水平和血浆中的EBVDNA载量。结果普查广西苍梧县城自然人群共1233人，其中80人IgA／VCA抗体阳性，阳性率为6．0％。对其中的72例IgA／VCA抗体阳性人群和20未治疗的鼻咽癌患者进行了LMP2特异性细胞免疫和EBVDNA检测，随着抗体滴度的升高，LMP2特异性细胞免疫反应水平降低，而血浆中EBVDNA拷贝数有随IgA／VCA抗体滴度升高而增加的趋势。结论EBV—LMP2特异性细胞免疫水平和EBVDNA拷贝数可能与IgA／VCA抗体滴度相关。

表达EBV LMP2的小鼠骨髓间充质干细胞免疫原性初讨

目的贴壁法分离小鼠骨髓间充质干细胞(MSCs),观察表达EBV LMP2蛋白的小鼠MSCs(MSCs-LMP2)体内诱导特异性CTL应答效果。方法贴壁培养法分离BALB/c小鼠MSCs,流式细胞术鉴定MSCs纯度。利用携带EBV LMP2基因的非复制重组5型腺病毒(Ad5-LMP2)感染已鉴定小鼠MSCs,制备表达EBV LMP2蛋白的MSCs-LMP2。使用MSCs-LMP2肌内注射的方式免疫BALB/c小鼠,每次免疫剂量6×105个细胞,免疫间隔为1周,连续免疫5次。末次免疫后1周分离小鼠脾淋巴细胞,Elispot方法检测EBV LMP2特异性CTL应答。结果贴壁培养法分离、纯化的小鼠骨髓MSCs纯度可达91.8%;Ad5-LMP2感染的MSCs能很好表达LMP2蛋白,形成MSCs-LMP2,MSCs-LMP2免疫同种小鼠后,在1×106个小鼠脾淋巴细胞中,诱导产生的LMP2特异性CTL能达到323个。结论贴壁培养法能够获得纯度较高的小鼠骨髓MSCs;LMP2修饰的MSCs(MSCs-LMP2)能诱导同种小鼠产生特异性CTL应答。

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.

EB病毒阳性肺癌组织中LMP-1、p53、Bcl-2及MMP-9表达

[目的]检测EB病毒(EBV)阳性肺癌组织中EBV潜伏感染膜蛋白-1(LMP-1)、p53、Bcl-2及基质金属蛋白酶-9(MMP-9)表达情况,分析它们与肺癌发生发展的关系。[方法]采用原位杂交法检测108例肺癌组织和22例癌旁正常肺组织中EBV编码的小RNA-1(EBV encoded small RNA-1,EBER-1)。免疫组织化学的方法检测EBER-1阳性和阴性肺癌组织中LMP-1、p53、Bcl-2及MMP-9的表达。[结果]108例肺癌组织中EBER-1阳性36例,阳性率33.3%;22例癌旁正常肺组织中EBER-1阳性1例,阳性率4.5%,差异有统计学意义(P<0.01)。EBER-1阳性和阴性的肺癌组织中LMP-1阳性率分别为11.1%和4.2%,差异无统计学意义(P>0.05);在EBER-1阳性肺癌组织中p53、Bcl-2的平均面积(AA)和积分光密度(IOD)均高于阴性组,差异有统计学意义。在EBER-1阳性肺癌组织中MMP-9AA和IOD均高于阴性组,但差异无统计学意义(P>0.05)。[结论]EBV感染可能通过影响LMP-1、Bcl-2、p53和MMP-9在肺癌组织中的表达,进而在肺癌的发生、发展和转移中发挥作用。

人IL-2与EB病毒LMP1融合基因重组BCG的构建与表达

目的构建并鉴定人IL-2与EB病毒LMP1融合基因重组BCG(卡介苗)。方法利用RT-PCR从EB病毒阳性的B95-8细胞中获得去除致癌部分的LMP1基因,与扩增的IL-2通过overlap-PCR扩增获得融合基因。将融合基因连接到穿梭表达质粒pMV261,进行酶切、PCR及测序鉴定。重组质粒经电转导入BCG,通过Western blot鉴定目标抗原在rBCG中的表达。结果 PCR扩增获得453bp IL-2和1 225bp LMP1,利用overlap-PCR获得1 623bp的融合基因,通过电转仪将重组质粒导入BCG感受态细胞,构建的重组质粒能够在BCG中表达相对分子质量为5.7×104的目的蛋白,Western blot显示该蛋白能被兔IL-2单抗及鼠LMP1单抗识别。结论构建的rBCG能稳定表达融合蛋白,为rBCG的抗肿瘤作用和抗肿瘤疫苗的研发奠定了基础。