HPV16 E6/E7与结核杆菌HSP70 N端重组DNA免疫对BALB/C小鼠免疫应答的影响

目的 :探讨HPV16 (人乳头状瘤病毒 16 )E6 /E7(原癌基因E6 /E7型 )以及与HSP70N端重组DNA免疫小鼠后 ,小鼠体内免疫应答的变化。方法 :以HPV16E6 /E7为基础的DNA疫苗免疫小鼠 ,并经酶联免疫吸附实验 (ELISA)及逆转录聚合酶链反应 (RT PCR)技术检测小鼠脾淋巴细胞产生的TH1/TH型细胞因子及血清抗体。结果 :HPV16DNA疫苗免疫组小鼠 ,其脾淋巴细胞IL 2及IFNγ的分泌量明显较对照组增加 (P <0 0 1) ;HPV16E6 /E7与HSP70N端重组后疫苗免疫小鼠 ,E6 HSP70N组产生的IFNγ量比重组前高 (P <0 0 1) ,其余重组组产生的IFNγ量及所有重组组产生的IL 2均较重组前低 (P <0 0 1) ,而各重组组产生的抗体水平较重组前低 (P <0 0 5 )或无明显差异 (P >0 0 5 )。结论 :以HPV16E6 /E7为基础的DNA疫苗能增强小鼠的细胞免疫反应 ,对体液免疫几乎无影响。HPV16E6 /E7与结核杆菌HSP70N端重组后的疫苗与重组前比较不增强细胞免疫反应 ,不影响体液免疫反应。     

HPV_(16)L1-E7重组病毒及其重组质粒免疫后小鼠抗体水平动态变化的研究

目的 :研究人乳头瘤病毒 16型L1 E7重组腺病毒 (rAd5HPV16 L1 E7virus)及其重组质粒 (rAd5HPV16 L1 E7plasmid)经不同途径免疫后小鼠抗体水平的动态变化。方法 :用 2 93细胞扩增rAd5HPV16 L1 E7重组病毒 ,同时制备rAd5HPV16 L1 E7重组质粒 ,分别以肌肉注射、腹腔注射、滴鼻和灌胃途径免疫小鼠 ,采用ELISA法测定其血清IgG抗体水平。结果 :rAd5HPV16 L1 E7重组病毒及其重组质粒采用不同途径免疫的BALB/c小鼠血清IgG抗体水平均高于对照组 (P <0 .0 5 ) ,其中以肌注组产生抗体量最多 ,并且出现最早。结论 :rAd5HPV16 L1 E7重组病毒及其重组质粒均能刺激B细胞产生抗体介导的免疫应答 ,免疫途径不同 ,抗体峰值的出现时间亦不同 ,测定抗体的最佳时间亦不同。     

HER-2/neu胞外区的基因工程表达及其免疫避孕作用

目的:研究HER-2/neu胞外区部分片段重组蛋白疫苗在免疫避孕中的作用。方法:构建原核表达重组体pET-hECDu,在大肠杆菌中表达人HER-2/neu胞外区部分片段,经Hi-Trap亲和层析柱纯化得到重组蛋白(hECDu),以ELISA法检测其结合活性后免疫雌性小鼠。用ELISA法检测抗HER-2/neu体液免疫应答,3H-TdR掺入法检测细胞免疫应答。结果:经原核表达系统成功纯化得到与理论分子量77 ku(1 u=1 Da)相符的hECDu,并证实其可与抗HER-2/neu胞外区特异性单克隆抗体结合。该重组蛋白疫苗可诱导雌鼠产生较高水平体液及细胞免疫应答。其生育力(平均产仔数4.0±1.8)与对照组(7.8±1.3)相比明显降低,但免疫鼠产后4 d子宫及卵巢均无明显病理改变。结论:经原核表达系统成功纯化得到HER-2/neu胞外区部分片段重组蛋白,该蛋白疫苗可在小鼠中诱导产生免疫避孕效应。     

治疗性HPV多肽疫苗的研究进展

高危型人乳头瘤病毒(HPV)的感染与宫颈癌密切相关,在子宫颈癌中HPV DNA的检出率达99%以上.高危型HPV E6和E7的表达是维持细胞转化所必需,被认为是HPV的原癌基因,表达于肿瘤进展的各个时期,是至今被选择作为治疗性疫苗理想的特异性靶抗原,这些原癌蛋白可以影响肿瘤特异性免疫应答.HPV疫苗用以预防和治疗宫颈癌已成为近年来的研究热点,本文就治疗性多肽疫苗的研究方法及现状做一综述.     

人乳头状瘤病毒16型多肽疫苗的制备及体内外效应观察

目的 探讨人乳头状瘤病毒(HPV)16型多肽疫苗的制备,并观察HPV16多肽疫苗的体内外效应.方法 (1)针对抗原加工相关转运子(TAP)设计HPV16 E7蛋白的主要组织相容性复合物Ⅰ类分子(MHC-I)的抗原结合表位,利用生物信息学分析平台筛选出一致性较高、特异性及亲和力较强的HPV16 E7多肽作为研究对象制备HPV16多肽疫苗用于以下研究,本研究共筛选出3段多肽,分别命名为E7Pa、E7Pb、E7Pc.(2)C57BL/6小鼠注射鼠肺上皮细胞株TC-1细胞(为鼠源性的HPV16阳性的肿瘤细胞株)后,采用等额抽取的随机方法分为5组,ETPa+二核苷胞嘧啶(CpG)、E7Pb+CpG、E7Pc+CpG[均为实验组,分别加入终浓度为50μg/ml的E7Pa、E7Pb、E7Pc和终浓度为12 mg/L的刀豆蛋白(ConA)]、CpG(为阳性对照,加入终浓度为12 mg/L 的Con A)和空白对照组(不做任何处理).采用四甲基偶氮唑蓝(MTT)比色法检测各组作用不同时间后小鼠脾T淋巴细胞的体外增殖效应,乳酸脱氢酶(LDH)释放法检测小鼠脾T淋巴细胞在不同效靶比下的体外细胞毒T淋巴细胞(CTL)活性,实时荧光定量RT-PER技术检测小鼠肿瘤组织中γ干扰素(IFN-γ)、白细胞介素2(IL-2)mRNA的表达水平,酶联免疫吸附试验(ELISA)检测小鼠外周血中IFN-γ、IL-2的表达水平,通过定期测量比较各组小鼠接种HPV16多肽疫苗后体内肿瘤体积的变化.结果 (1)本研究筛选出了一致性较高、特异性及亲和力较强的3段HPV16 E7多肽作为研究对象制备HPV16多肽疫苗,分别命名为E7Pa、E7Pb、E7Pc.(2)MTT比色法检测显示,在接种疫苗24、48、72、96 h后,以E7Pa+CpG组的增殖效应最明显,其细胞增殖率分别为(131±32)%、(302±15)%、(552±28)%、(731±24)%,明显高于空白对照组的(72±15)%、(120±57)%、(176±41)%、(288±29)%(P<0.01);E7Pb+CpG、ETPc+CpG、CpG组的细胞增殖率也均明显高于空白对照组(P<0.05);但E7Pb+CpG、E7Pc+CpG、CpG组间比较,差异则无统计学意义(P>0.05).LDH释放法榆测显示,效靶比为100:1时,E7Pa+CpG、E7Pb+CpG、E7Pc+CpG、CpG和空白对照组CTL 活性分别为(85.9±3.0)%、(55.9±2.5)%、(60.2±1.5)%、(41.0±1.7)%和(4.1±1.0)%,E7Pa+CpG组与空白对照组比较,差异有统计学意义(P<0.01);E7Pb+CpG、E7Pc+CpG、CpG组分别与空白对照组比较,差异也有统计学意义(P<0.05);但E7Pb+CpG、E7Pc+CpG、CpG组间比较,差异则无统计学意义(P>0.05).在肿瘤组织及外周血中,小鼠IFN-γ、IL-2的表达水平,E7Pa+CpG组与空白对照组比较,差异均有统计学意义(P<0.01);E7Pb+CpG、E7Pc+CpG和CpG组分别与空白对照组比较,差异也均有统计学意义(P<0.05);但E7Pb+CpG、E7Pc+CpG、CpG组问比较,差异则无统计学意义(P>0.05).小鼠体内的肿瘤体积,各实验组肿瘤生长均明显被抑制,接种后第60大,E7Pa+CpG组与空白对照组比较,差异有统计学意义(P<0.01);E7Pb+CpG、E7Pc+CpG和CpG组分别与空白对照组比较,差异也均有统计学意义(P<0.05);但 E7Pb+CpG、E7Pc+CpG、CpG组间比较,差异则无统计学意义(P>0.05).结论 在动物模型中,针对TAP筛选的HPV16 E7多肽联合CpG制备的HPV16多肽疫苗,可以有效治疗HPV16 E7阳性的肿瘤.     

HPV58型E2蛋白表达纯化与多克隆抗体制备

目的:表达纯化重组人乳头瘤病毒(human papillomavirus,HPV)58型E2蛋白,制备多克隆抗体。方法:构建重组质粒p ET-28b-E2,转化至BL21(DE3)p Lys S中诱导表达,包涵体洗涤后经镍柱亲和层析分离得到纯化目的蛋白。用纯化的重组E2蛋白免疫新西兰白兔,制备兔抗HPV58型E2蛋白多克隆抗体,ELISA分析多克隆抗体的效价,免疫印迹检测抗体的特异性。结果:表达纯化了重组HPV58型E2蛋白,制备了高滴度和高特异性的多克隆抗体。结论:制备的多克隆抗体可用于对HPV58型E2蛋白进行精细B细胞线性表位鉴定。     

治疗性HPV多肽疫苗的研究进展

高危型人乳头瘤病毒(HPV)的感染与宫颈癌密切相关，在子宫颈癌中HPV DNA的检出率达99％以上。高危型HPVE6和E7的表达是维持细胞转化所必需，被认为是HPV的原癌基因，表达于肿瘤进展的各个时期，是至今被选择作为治疗性疫苗理想的特异性靶抗原，这些原癌蛋白可以影响肿瘤特异性免疫应答。HPV疫苗用以预防和治疗宫颈癌已成为近年来的研究热点，本文就治疗性多肽疫苗的研究方法及现状做一综述。     

HPV16 E6E7癌蛋白通过TWEAK/Fn14信号途径对细胞增殖和凋亡的影响

目的:探讨HPV16 E6E7对TWEAK/Fn14信号途径及细胞增殖和凋亡的影响。方法:制备表达HPV16 E6E7的逆转录病毒,感染原代角质形成细胞。用流式细胞仪分析可诱导纤维母细胞生长因子14(Fn14)表达,Real-time RT-PCR和Western blot法检测调解活化正常T细胞表达和分泌的趋化因子(RANTES)、单核细胞趋化蛋白-1(MCP-1)、干扰素-γ诱导蛋白10水平(IP10)和肿瘤坏死因子(TNF)受体相关因子(TRAF2)表达,生物化学法检测Ras GTpase活性,TUNEL检测细胞的凋亡,MTT法检测细胞增殖。结果:表达HPV16 E6E7的逆转录病毒感染角质形成细胞后,Fn14高表达,RANTES、MCP-1、IP10和TRAF2表达增加,Ras GTpase活性升高。与未感染表达HPV16 E6E7逆转录病毒的细胞比较,表达E6E7感染的角质形成细胞对TNF-α刺激不敏感,细胞增殖显著增加(P0.05)。结论:表达HPV16 E6E7细胞激活TWEAK/Fn14信号途径,从而上调RANTES和TRAF2表达,导致细胞增殖,这可能是高危型HPV的一个致病机制。     

基于抗原表位分析的人乳头瘤病毒16L1壳蛋白基因克隆及表达纯化

目的:选择人乳头瘤病毒(HPV)16L1抗原性最强的一段抗原表位编码区,构建原核表达载体并利用大肠杆菌表达抗原蛋白,为HPV疫苗的研究及宫颈癌的诊断奠定基础。方法:借助DNAstar软件分析HPV16L1的CDS区,选择抗原性最强的一段抗原表位编码区,提取HPV16L1编码区的DNA。目的 DNA和p ET32a质粒分别进行双酶切,连接并转化DH5α大肠杆菌,筛选阳性克隆后转化BL21大肠杆菌,诱导产生HPV16L1重组蛋白,通过市售抗体对重组蛋白的抗原特异性进行鉴定。结果:利用DNAstar软件,选择了抗原表位较富集的碱基序列919~1314bp区段,按常规制备重组蛋白的方法成功制备HPV16L1重组蛋白。抗体鉴定表明,制备的重组蛋白抗原特异性良好。结论:有针对性地制备HPV16L1重组蛋白,可避免其他抗原表位的干扰。经鉴定,HPV16L1重组蛋白抗原特异性良好,将为HPV疫苗的研究及宫颈癌的诊断奠定基础。     

080树突状细胞为基础的疫苗治疗宫颈癌:一项15例患者个体化治疗的临床实验结果

人类乳头瘤病毒(HPV)16,18是宫颈癌中最常见的基因型.HPV16,18的癌基因E6,E7的表达产物是宫颈癌肿瘤细胞的永恒表型.由于宫颈癌肿瘤细胞有强阳性病毒抗原表达,故认为宫颈癌适合进行免疫治疗.研究中15例Ⅳ期宫颈癌患者经PCR-ELASA分析,12例检测出HPV16,3例检测出HPV18.将自体单核细胞衍生而来的树突状细胞(DC)体外加载重组HPV16E7或HPV18E7癌蛋白制成疫苗,对患者按个体化方案进行至少1次免疫接种.治疗前后监测接种的安全性、毒副作用、患者的临床反应、血清学反应(包括血清SCC、CEA水平)及免疫反应(ELISA分析检测抗HPV16,18的E6,E7抗原的抗体水平变化及抗原特异性T分析Th1增殖反应和CTL反应).     

A DNA vaccine constructed with human papillomavirus type 16 (HPV16) E7 and E6 genes induced specific immune responses

OBJECTIVE: Cervical cancer is found highly associated with human papillomaviruses type 16 (HPV16). HPV16 E6 and E7 oncogenes are important transforming genes which have become the main focus of anti-cervical cancer therapy. In this study, a recombinant DNA vaccine candidate, termed HPV16-DNA-E6E7, constructed with HPV16 E7 and E6 genes was generated and used to against HPV16-induced tumors. METHODS: We inserted an E7 DNA fragment into E6 gene to produce a recombinant gene (E6E7-DNA). The E6E7-DNA gene was inserted into a mammalian expression vector, pcDNA 3.1+, to construct the DNA vaccine candidate. Animals (C57BL/6 mice) were immunized with the vaccine candidate with various concentrations (50 microg, 100 microg or 200 microg, respectively), and cytotoxicity measurement and tumor protection assay were carried out to examine the immunological effects of the vaccine candidate. RESULTS: Immunization of with HPV16-E6E7-DNA induced HPV16-specific immune response and also conveyed protection against TC-1 induced tumor in vivo. A survival rate (90%) after 45 days of tumor challenge was observed. The animals injected with a higher dosage of the vaccine (200 microg) exhibited prolonged survival duration of more than 55 days. No transforming activity of the vaccine candidate was detected, as determined by focus formation and degradation of endogenous p53. CONCLUSION: Our results demonstrated that the HPV16-E6E7-DNA compound might become a candidate for HPV16 precautionary and immunotherapy.     

含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1-E7AS的构建及鉴定

目的:构建含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1- E7AS并鉴定,探讨腺相关病毒介导的反义E7基因技术用于治疗早期宫颈癌的可能性。方法:使用RT -PCR法扩增全长HPV16型E7基因,利用基因重组法将目的片段反向插入腺相关病毒骨架质粒pUF1并酶切鉴定。结果:RT PCR法扩增全长315bp的HPV16型E7基因,装入pGEM -Teasy载体进行测序,经NCBI数据库blast检索为100%的符合,经基因重组获得含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1 E7AS,AccⅠ和KpnⅠ双酶切鉴定pUF1 E7AS。结论:含HPV16型反义E7基因的腺相关病毒骨架质粒pUF1 E7AS可成功构建。     

Human papillomavirus type 16 E7 peptide(38-61) linked with an immunoglobulin G fragment provides protective immunity in mice

OBJECTIVE: To explore whether the recombinant protein (Human papillomavirus (HPV) type16 E7 peptide(38-61) linked with an immunoglobulin G fragment) will generate protective immunity in mouse model. METHODS: In our study, we combined the HPV16 E7 peptide(38-61) with a murine IgG heavy chain constant region to construct a chimeric protein compound, which was highly expressed as inclusion bodies in a bacterial expression system with Escherichia coli. The purified chimeric protein was injected into C57BL/6 mice and the efficiency of the chimeric vaccine candidate was evaluated by antibody response assay, T cell proliferation assay, CTL assay, tumor challenge assay and therapeutic experiment. RESULTS: The chimeric vaccine candidate was able to induce anti-HPV antibodies as well as to elicit HPV16 E7-specific CTLs and T cell proliferation in a pre-clinical mouse model. It was also able to effectively protect mice against the challenge of HPV16-positive tumor cells, and to eradicate HPV16-expressing tumors in mice. CONCLUSIONS: The chimeric protein vaccine can induce E7-specific immune responses and protect mice against challenge of HPV16-positive tumor, even eradicate developed tumor. The results indicated a possibility to use the chimeric protein vaccine to protect human against HPV infection.     

人乳头状瘤病毒16型E7基因重组腺相关病毒载体的构建及E7 mRNA和蛋白在真核细胞中的表达

目的构建含人乳头状瘤病毒16型E7(HPV16E7)基因的重组腺相关病毒(rAAV)载体,并验证HPV16E7mRNA和蛋白在真核细胞中的表达。方法将含腺相关病毒(AAV)末端反向重复序列及HPV16E7基因的重组质粒pAAVMCSE7、含rep/cap基因的质粒pAAVRC及辅助质粒pAAVhelper共同转染胚胎肾细胞系HEK293细胞,回收、纯化病毒颗粒并鉴定,斑点杂交法测定病毒滴度,RTPCR技术、蛋白印迹法验证HPV16E7mRNA和蛋白在真核细胞中的表达。结果电镜鉴定结果显示真核细胞中有病毒颗粒存在,斑点杂交法测定病毒滴度为1×1011/ml。含HPV16E7基因的rAAV载体转染真核细胞后,在细胞内可检测到HPV16E7mRNA和蛋白的表达。结论本实验成功构建了含HPV16E7基因的rAAV载体,经验证HPV16E7mRNA和蛋白在真核细胞内有表达。     

HPV16/18 E7-pulsed dendritic cell vaccination in cervical cancer patients with recurrent disease refractory to standard treatment modalities

OBJECTIVE: To evaluate the potential of human papillomavirus (HPV) type 16 and 18 E7 antigen-loaded autologous dendritic cells (DC) as a therapeutic cellular vaccine in a case series of cervical cancer patients harboring recurrent/metastatic disease refractory to standard treatment modalities. METHODS: Autologous monocyte-derived DC were pulsed with recombinant HPV16 E7 or HPV18 E7 oncoproteins and administered to 4 cervical cancer patients. Vaccinations were followed by subcutaneous administration twice daily of low doses of human recombinant interleukin-2 (1 x 10(6) IU/m2) from day 3 to day 7. Safety, toxicity, delayed type hypersensitivity reactions (DTH), clinical responses, and induction of serological and cellular immunity against HPV16/18 E7 were monitored. RESULTS: The vaccine was well-tolerated in all patients and no local or systemic side effects or toxicity were recorded. Three out of four patients were found to be significantly immunocompromised before starting the vaccination treatment, as assessed by DTH with a panel of recall antigens. Specific humoral and cellular CD4+ T cell responses to the E7 vaccine were detected in 2 patients, as detected by ELISA and by IFN-gamma ELISpot assays, respectively. Increased numbers of E7-specific IFN-gamma secreting CD8+ T cells were detected in all patients after vaccination. Swelling and induration (i.e., a positive DTH response) to the intradermal injection of HPV E7 oncoprotein and/or irradiated autologous tumor cells were detected in two patients after six vaccinations. No objective clinical responses were observed. However, both patients who developed a positive DTH to the vaccine experienced a slow tumor progression (i.e., 13 months survival) while DTH unresponsive patients died within 5 months from the beginning of therapy. CONCLUSIONS: Autologous DC pulsed with HPV16/18 E7 proteins can induce systemic B and T cell responses in patients unresponsive to standard treatment modalities. However, treatment-induced immunosuppression may impose severe limitations on the efficacy of active vaccination strategies in late stage cervical cancer patients. DC-based vaccination trials are warranted in immunocompetent cervical cancer patients with early stage disease and/or limited tumor burden, and at significant risk for tumor recurrence or disease progression.     

Prime-boost vaccination strategy in women with high-grade, noncervical anogenital intraepithelial neoplasia: clinical results from a multicenter phase II trial

The objective of this study was to determine the clinical effectiveness of a prime-boost human papillomavirus (HPV) vaccine regimen. A nonrandomized phase II prime-boost vaccine trial was conducted. Women with biopsy-proven anogenital intraepithelial neoplasia (AGIN) 3 were vaccinated with three doses of a recombinant fusion protein comprising HPV 16, E6/E7/L2 (TA-CIN) followed by one dose of a recombinant vaccinia virus encoding HPV 16 and 18 E6/E7 (TA-HPV). Clinical responses were evaluated by serial photographs, symptomatology, and biopsies before and after vaccination. Twenty-nine women were vaccinated; 27 with vulval intraepithelial neoplasia 3 and 2 with vaginal intraepithelial neoplasia grade 3. Clinical responses were seen in five women (17%), with one complete and five partial responses. Fifteen women (62%) had symptomatic improvement. No serious adverse effects were recorded. This is the first trial of a prime-boost vaccination regimen using heterologous HPV vaccines (TA-CIN followed by TA-HPV) in the management of AGIN. Since the prime-boost approach in this cohort offered no significant advantages over single TA-HPV vaccination, there are no further studies planned using this protocol. Future studies are warranted to define responders to immunotherapy.     

腺病毒伴随病毒表达载体表达人乳头状瘤病毒16型E6/E7基因的构建及应用

目的　为了了解人乳头状瘤病毒 (Humanpapillomavirus ,HPV) 1 6型的E6 /E7基因在细胞恶性转化中所起的作用 ,利用腺病毒伴随病毒载体 (AAVHelper -FreeSystem)构建和表达人乳头状瘤病毒 1 6型E6 /E7基因。方法　在pLXSN1 6E6E7质粒中经PCR扩增回收HPV 1 6E6E7基因片段 ,连接于T载体上进行测序 ,将正确的HPV 1 6E6E7插入pAAV -IRES -hrGFP质粒 ,协同pAAV -RC质粒和pHelper质粒共转染HEK 2 93细胞 ,包装表达HPV 1 6E6E7基因的重组腺病毒伴随病毒 ,收获病毒 ,并检测病毒的感染效率。结果　在包装细胞系HEK 2 93细胞中能形成较高感染效率的腺病毒伴随病毒 ,激光共聚焦检测可发现HEK 2 93细胞内有绿色荧光蛋白表达 ,HEK 2 93细胞经PCR可扩增出特异性的HPV 1 6E6E7基因片段 ,经流式细胞仪检测重组病毒的感染效率为71 3%。结论　携带人乳头状瘤病毒 1 6型E6E7基因的腺病毒伴随病毒可感染细胞 ,并在细胞内表达 ,可望用于宫颈癌病因学的研究     

反义HPV16 E6/E7-EGFP重组质粒的构建及其对宫颈癌SiHa细胞的促凋亡作用

目的:构建HPV16早期基因E6/E7的反义重组质粒,探讨其对SiHa细胞的促凋亡作用。方法:将HPV16E6/E7基因片段反向克隆于真核表达载体pEGFP-C1并转染SiHa细胞,用RT-PCR方法检测转染后SiHa细胞E6、E7基因mRNA的表达,West-ernblot方法检测转染后E6/E7蛋白的表达,流式细胞仪检测转染后细胞的凋亡率。结果:成功构建携带HPV16E6/E7基因反义片段的真核表达载体,转染该质粒后,SiHa细胞E6、E7基因的mRNA和蛋白均明显下调;转染后细胞凋亡率为(59.3±11.3)%,明显高于转染空载体组[(9.4±1.8)%]和未转染组[(2.1±0.4)%](P<0.05)。结论:反义HPV16E6/E7基因可下调宫颈癌细胞中E6/E7癌基因的表达,诱导宫颈癌细胞凋亡,为宫颈癌的基因治疗提供了实验依据。     

Enhancing immunogenicity of HPV16 E7 DNA vaccine by conjugating codon-optimized GM-CSF to HPV16 E7 DNA

ObjectiveTo generate immunity against human papillomavirus (HPV), the use of a recombinant DNA vaccine to carry an appropriate target gene is a promising and cost-effective approach. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immunomodulatory cytokine that enhances the efficacy of vaccines by promoting the development and prolongation of humoral and cellular immunity. In this study, we linked codon-optimized GM-CSF (cGM-CSF) to the HPV16 E7 sequence as fused protein and evaluated the immunogenic potential of this DNA vaccine.Materials and methodsWe have demonstrated that cGM-CSF enhanced immunity against tumor challenges by generating and promoting the proliferation of HPV16 E7-specific CD8+ T cells, which secrete IFN-γ in the murine model. In this study, we aimed to evaluate the immunogenic potential of DNA vaccine that constructed by linking codon-optimized GM-CSF to HPV16 E7 sequence in the animal model. We study the half-life of RNA decay and cellular location of HPV16 E7 by Q-PCR and Western blot. We also assess immune response in the animal model by flow cytometry and ELISA.ResultsThe cGM–CSF–E7 sequence increased and extended the expression of E7 mRNA, in comparison with the E7 sequence alone. Mice vaccinated with the cGM–CSF–E7 DNA vaccine exhibited a slower rate of tumor growth than those vaccinated with the unconjugated E7 DNA vaccine. We also found that the CD4 and CD8+ T cells from these mice showed strong secretion of IFN-γ.ConclusionThrough in vivo antibody depletion experiments, we demonstrated that both CD4+ and CD8+ T cells play an important role in the suppression of tumor growth.