改造的人乳头瘤病毒16型E6/E7融合蛋白疫苗抗肺癌作用的实验研究

目的 研究经改造的HPV16 E6／E7蛋白质疫苗(HPV16 mE6Δ／mE7)对表达HPV16 E6和E7的小鼠肺癌细胞的体内生长抑制作用，为肺癌免疫治疗提供新线索。方法 预防性实验：对C57BL／6小鼠先进行免疫，然后接种表达HPV16E6和E7癌蛋白的TC-1肿瘤细胞；第33天，对未出瘤小鼠进行第二次大剂量肿瘤细胞攻击。治疗性实验：C57BL／6小鼠先接种TC-1细胞，然后进行两次免疫治疗，再对末出瘤小鼠进行第二次大剂量TC-1细胞攻击实验。计算各组肿瘤发生率、肿瘤体积。采用MTT方法检测淋巴细胞增殖反应。结果预防性实验：在100天的观察时间内，免疫组小鼠可以有效抵抗第一、二次肿瘤细胞攻击，所有小鼠均未见肿瘤发生；而PBS组及单纯佐剂组小鼠在第一次肿瘤细胞接种后6～12天内均有肿瘤形成，30天内因肿瘤负荷出现衰竭。在治疗性实验中，免疫组、PBS组、单纯佐剂组小鼠成瘤率分别为20％、90％、60％；第二次攻击实验中，免疫组仍有87．5％的小鼠无肿瘤生长，而其他两组动物在4～6天内均有肿瘤形成。MTT实验表明经mE6Δ／mE7免疫的小鼠脾淋巴细胞增殖反应明显强于对照组。结论 经改造的HPV16型mE6Δ／mE7蛋白质疫苗对于实验动物肺癌有较好的抑制作用，为HPV16相关肺癌的免疫治疗提供了实验数据。     

HPV16型重组疫苗的构建及其抗肿瘤效果

背景与目的大量研究表明,宫颈癌的发生与高危型人乳头状瘤病毒(humanpapillomavirus,HPV)的感染密切相关,其中以HPV16为主。本研究以非复制型重组痘苗病毒为载体,构建共表达HPV16L1、L2、E67蛋白的重组非复制型痘苗病毒,并检测其免疫效果。方法以痘苗病毒为载体,利用同源重组技术构建共表达HPV16L1、L2、E67基因的重组痘苗病毒NTVJE67CKL1L2。用该病毒免疫C57BL/6小鼠后,检测其特异性抗体和特异性的细胞毒性T淋巴细胞(cytotoxicTlymphocyte,CTL);以TC-1肿瘤细胞攻击经免疫后的小鼠,观察其免疫保护效果。结果经斑点杂交结果显示重组病毒基因组中有L1、L2、E6、E7基因插入;经Westernblot检测,重组病毒能稳定表达HPV16L1、L2、E67蛋白。动物实验结果表明,NTVJE67CKL1L2在C57BL/6小鼠体内可诱发L1、L2、E67特异性抗体产生;可诱发产生针对TC-1细胞的特异性的CTL反应;加强免疫后的小鼠能耐受1×104个TC-1肿瘤细胞的攻击。结论非复制型重组痘苗病毒NTVJE67CKL1L2可以作为对HPV16相关肿瘤及其癌前病变进行免疫治疗的候选疫苗。     

携带HPV16 E7 shRNA和IL-12基因的重组短双歧杆菌抗小鼠宫颈癌移植瘤的疗效和安全性

目的：评价口服携带HPV16 E7 shRNA和IL-12基因的重组短双歧杆菌在小鼠体内抗宫颈癌移植瘤的效果。方法：将p MG36e-E7 shRNA、pMG36e-mIL-12D质粒分别转化短双歧杆菌,经筛选鉴定并扩增获得携带HPV16 E7 shRNA和IL-12基因的重组短双歧杆菌。通过小鼠皮下宫颈癌细胞移植建立荷瘤小鼠模型。口服重组短双歧杆菌1、7 d后,检测小鼠主要器官（心、肝、脾、肺、肾）和肿瘤组织匀浆液或血清在PYG培养基中形成的菌落数量,评价短双歧杆菌的肿瘤靶向性,以小鼠体内肿瘤生长曲线评估重组短双歧杆菌的抗肿瘤效果,通过主要器官切片H-E染色和检测荷瘤小鼠血清相关细胞因子水平评价口服重组短双歧杆菌的安全性。结果：成功制备重组短双歧杆菌和宫颈癌TC-1细胞移植瘤小鼠。7 d后,移植瘤组织匀浆液和血清的菌落数量证实短双歧杆菌具有靶向体内瘤组织的定殖能力,口服重组短双歧杆菌明显抑制荷瘤小鼠的肿瘤生长（P<0.05或P<0.01）,但联合使用携带HPV16 E7 shRNA和IL-12基因的重组双歧杆菌的肿瘤抑制率与单独使用的并无显著差异,治疗后未见对荷瘤小鼠主要...     

肿瘤基因工程纳米疫苗NL(MH)抗肿瘤复发的实验研究

目的：在小鼠体内观察纳米疫苗NL（MH）抗肿瘤复发作用。方法：以PBS、空白脂质体、MH、MH／NL、NL（MH）免疫C57BL／6小鼠3次后，IFN-γ ELISPOT和LDH杀伤实验检测纳米疫苗激活小鼠特异性细胞免疫反应的情况；以肿瘤攻击实验和肿瘤切除后复发实验来评价纳米疫苗预防肿瘤复发作用。结果：与对照组相比，NL（MH）组小鼠脾淋巴细胞中分泌IFN-γ的T细胞数量明显增多（P〈0．05），CTL对B16-MAGE3细胞具有显著的特异性杀伤作用；在肿瘤攻击实验中，NL（MH）组B16-MAGE3肿瘤成瘤时间长、成瘤率低；肿瘤切除后，NL（MH）组B16-MAGE3肿瘤复发时间延迟，复发率明显降低。结论：NL（MH）能够刺激机体产生强烈的MAGE3特异性的细胞免疫反应，对表达MAGE3的肿瘤细胞具有显著的杀伤作用，能有效预防B16-MAGE3切除后复发。     

树突状细胞疫苗的制备及其体外诱导细胞毒性T淋巴细胞对宫颈癌CaSki细胞的杀伤作用

目的:制备树突状细胞(dendritic cells,DCs)疫苗并观察其在体外诱导细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTLs)对宫颈癌CaSki细胞的杀伤效应。方法:分离培养小鼠未成熟DCs,FCM检测小鼠未成熟DCs表面标志物CD40、CD86、主要组织相容性复合体-Ⅱ(major histocompatibility complex-Ⅱ,MHC-Ⅱ)和CD11c;将已成功构建的携带人乳头状瘤病毒(human papillomavirus,HPV)16E6E7基因的重组腺病毒pAd-E6E7感染体外培养的小鼠未成熟DCs,提取CaSki细胞裂解物负载DCs,制备DC疫苗,激光共聚焦显微镜下观察pAd-E6E7感染的小鼠未成熟DCs绿色荧光蛋白表达,Western印迹法检测E6蛋白的表达;DCs疫苗诱导产生CTLs后与CaSki细胞共培养,CCK8(cell countingkit8)法检测其对CaSki细胞的杀伤效应。结果:成功分离培养了小鼠未成熟DCs,并制备获得了HPV16E6E7特异性DCs疫苗。DCs疫苗诱导产生的CTLs对CaSki细胞具有杀伤作用,pAd-E6E7感染组对CaSki的杀伤效应明显高于CaSki细胞裂解物负载组及未处理DCs组(P<0.05)。结论:以携带HPV16E6E7基因的重组腺病毒感染DCs制备基因修饰的DCs疫苗,具有诱导CTLs体外杀伤子宫颈癌CaSki细胞的作用。     

SLC基因修饰对肿瘤细胞疫苗免疫效果的初步研究

目的：构建次级淋巴组织趋化因子（secondary lymphoid—tissue chemokine，SLC）基因修饰的趋化型恶性黑色素瘤疫苗，并初步研究其免疫效果。方法：SLC基因转染小鼠黑色素瘤细胞B16，并用适当条件的丝裂霉素处理，制备趋化型肿瘤细胞疫苗。在体外，利用趋化小室法检测趋化型疫苗培养上清对小鼠脾淋巴细胞的趋化活性。在体内，用趋化型疫苗免疫小鼠，检测脾淋巴细胞的CTL活性，并观察疫苗免疫对B16移植瘤生长的影响。结果：筛选获得了SLC基因转染的小鼠黑色素瘤B16细胞，以100μg／mL丝裂霉素处理1.5h，制备趋化型肿瘤细胞疫苗。趋化型疫苗在体外培养48h后，其培养上清对淋巴细胞具有明显的趋化活性。与野生型疫苗相比，用趋化型疫苗免疫后，小鼠脾淋巴细胞的CTL活性更强，小鼠免疫后再次接种B16细胞，肿瘤的生长受到更明显的抑制。结论：成功构建了SLC基因修饰的趋化型黑色素瘤疫苗，并在体内外初步检测到该疫苗对抗肿瘤免疫的影响和对移植瘤生长的抑制，该疫苗有可能被应用于恶性黑色素瘤的免疫治疗。     

紫杉醇与肿瘤疫苗联合应用对小鼠Lewis肺癌皮下移植瘤的治疗效果

目的： 〖HT5"SS〗评价化疗药物紫杉醇联合应用肿瘤疫苗对小鼠Lewis肺癌皮下移植瘤的治疗效果。〖HT5W〗方法： 〖HT5"SS〗以编码有GMCSF的腺病毒感染Lewis肺癌细胞3LL,制备肿瘤疫苗3LL/GMCSF。以2×104个3LL瘤细胞皮下注射C57BL/6（H2b）小鼠腹股沟部,制备小鼠皮下移植瘤。检测体内、外应用紫杉醇对Lewis肺癌细胞3LL的杀伤敏感性。在小鼠Lewis肺癌皮下移植瘤体内首先以紫杉醇化疗,随后以肿瘤疫苗3LL/GMCSF免疫;或反之,首先以肿瘤疫苗免疫,随后以紫杉醇化疗,观察肿瘤生长和小鼠生存状况,以及检测小鼠体内对肿瘤的特异性杀伤效率和免疫应答记忆反应。〖HT5W〗结果〖HT5"SS〗： 紫杉醇体外作用于3LL肿瘤细胞,24 h后在浓度为100 nmol/L时可使32.10 %的3LL肿瘤细胞发生死亡;但体内注射紫杉醇（5、10和25 mg/kg）不能使所有3LL荷瘤小鼠肿瘤消退。体内使用紫杉醇后应用3LL/GMCSF肿瘤疫苗,70%的荷瘤小鼠发生显著的肿瘤消退,与单纯化疗的小鼠相比生存时间明显延长（70.0 vs 27.5 d）; 化疗后应用肿瘤疫苗诱导了体内对3LL的特异性杀伤,第3天体内杀伤率达41.35%;同时,存活小鼠能抵抗2×104 3LL肿瘤细胞的再次攻击。接种3LL/GMCSF肿瘤疫苗后应用紫杉醇化疗却不能使肿瘤消退。〖HT5W〗结论： 〖HT5"SS〗化疗后应用肿瘤疫苗免疫可诱导出抗原特异性免疫效应,使荷瘤小鼠肿瘤消退并延长生存时间,为临床开展化疗后的肿瘤疫苗主动免疫提供了实验依据。     

增殖病毒对肿瘤的治疗作用

目的：探讨ＨＥＲ２／ｎｅｕ原癌基因胞外区片段ＤＮＡ免疫诱导的特异性细胞免疫应答及其在体内的抗瘤效应。方法：克隆并构建人ＨＥＲ２／ｎｅｕ原癌基因及其胞外区片段表达载体,分别转染ＳＰ２／０细胞以制备杀伤靶细胞。将质粒ＤＮＡ免疫小鼠,观察其诱导的细胞免疫应答,同时分析免疫动物体内抗瘤效应。结果：体外获得稳定表达ＨＥＲ２／ｎｅｕ基因的ＳＰ２／０靶细胞。目的基因ＤＮＡ免疫后,免疫鼠脾细胞在体外可检测到特异性杀伤作用,体内肿瘤细胞接种后可发现肿瘤结节出现时间延迟,肿瘤生长缓慢。结论： ＨＥＲ２／ｎｅｕ原癌基因胞外区片段ＤＮＡ免疫可诱导出特异细胞免疫应答,并具有一定抗瘤效应。     

树突状细胞诱导抗裸鼠人肝癌移植瘤免疫

[目的]讨究树突状细胞（DC）体外诱导的抗肝癌细胞免疫能否预防裸鼠人肝癌移植癌发生及抑制课鼠人肝癌移植瘤生长。[方法]联合应用粒／巨噬细胞集落刺激因子（GM－CSF）及白介素－4（IL－4）直接从肝癌患者外周血中培养DC：以人肝癌细胞系HepG2肿瘤细胞的肿瘤抗原粗提物刺激DC；DC激活同源的T淋巴细胞产生细胞毒性T淋巴细胞（CYL）；以DC激活的CIL预防性接种于探鼠皮下观察其后接种的人肝癌细胞系HepG2移植瘤发生率；并观察该CTL对已接种裸鼠的人肝癌细胞系HepG2移植瘤生长的影响。[结果]DC诱导的工体外对HepG2肿瘤细胞具有高效而特异杀伤作用，在裸鼠体内不能预防探鼠人肝癌细胞系HepG2移植瘤发生，而且能抑制该移植瘤生长。[结论]经肝癌肿瘤相关抗原激活的肝癌患者外用血DC体外诱导的抗肿瘤细胞免疫在体内外均具有良好的抗肿瘤作用。提示DC作为一新概念上的抗肿瘤疫苗有可能在人肝癌的预防及治疗中发挥重要作用。     

cEGFR-rFc融合DNA疫苗抗小鼠黑素移植瘤的效果

目的:构建cEGFR-rFc融合DNA疫苗, 观察其对小鼠黑素移植瘤的抑制作用.方法:以异种同源鸡EGFR(chicken EGFR,cEGFR)膜外部分与兔疫球蛋白IgG的Fc段(rabbit IgG Fc,rFc)为基础构建真核表达载体pVAX1/cEGFR-rFc,脂质体法转染黑素瘤B16 细胞,免疫荧光法检测融合蛋白在B16细胞中的表达;以融合DNA疫苗免疫C57BL/6J小鼠,Western blotting法检测融合蛋白在小鼠体内的稳定表达.疫苗免疫小鼠接种B16黑素瘤细胞,14 d后处死部分小鼠,取出瘤体称重,计算抑瘤率;同时观察小鼠荷瘤后的生存率;ELASIA法检测DNA疫苗免疫小鼠血清抗EGFR抗体滴度,流式细胞仪测定小鼠脾细胞淋巴细胞亚群.结果:成功构建融合DNA疫苗pVAX1/ cEGFR-rFc,重组载体转染B16细胞后能检测到融合蛋白显著表达,疫苗免疫小鼠后能检测融合蛋白体内稳定表达.融合DNA疫苗能够延缓小鼠黑素移植瘤的生长,抑瘤率为54%(P<0.01);能延长荷瘤小鼠的生存期(疫苗组小鼠接种瘤细胞后30 d的生存率为40%);融合DNA疫苗诱发小鼠产生高滴度抗EGFR抗体(效价1∶1 000)和T细胞免疫(疫苗组小鼠脾脏CD8 T淋巴细胞数目显著增加, P<0.01).结论:cEGFR-rFc融合DNA疫苗能产生有效对抗黑素瘤B16细胞的免疫效应.     

Vaccination of full-length HPV16 E6 or E7 protein inhibits the growth of HPV16 associated tumors

Cervical cancer is the second most common cancer in women worldwide. Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Two HPV16 proteins, E6 and E7, are consistently expressed in tumor cells. Most therapeutic vaccines target one or both of these proteins. Taking the advantages of safety and no human leukocyte antigen restriction, protein vaccine has become the most popular form of HPV therapeutic vaccines. Here we demonstrate that immunization with full-length HPV16 E6 or E7 protein elicited specific immunological effect and inhibition of TC-1 cell growth using TC-1 mouse model. HPV16 E6 and E7 genes were cloned into pET-28a(+) and introduced into E. coli Rosetta. Expression of the genes was induced by IPTG. Proteins were purified by Ni-NTA agarose and they were detected by SDS-PAGE and Western blotting. C57BL/6 mice were vaccinated with 1.5 nmol HPV16 E6 or E7 protein. Then they were implanted with 1x10(5) TC-1 cells. No tumor was detected in any mouse vaccinated with E7 protein. Forty days later, the tumor-free mice and control mice were challenged with 2x10(5) TC-1 cells. All control mice developed tumors 6 days later, but E7 immunized mice were tumor free until 90 days. Tumor growth was slow in the E6 immunized mice, but 83% of the mice developed tumors and the survival percentage was not significantly different from the control. An adoptive immune model was used to demonstrate the therapeutic effect. Results showed that the development of TC-1 cells was obviously reduced by transfusion of T-cells but not serum from mice immunized with E7 protein. T-cells from E7 immunized mice also induced the lysis of TC-1 cells in the cytotoxic T lymphocyte assay. These findings show that immunization with HPV16 E6 or E7 protein was able to elicit specific protective immunity against TC-1 tumor growth.     

Immunotherapeutic Effects of Dendritic Cells Pulsed with a Coden-optimized HPV 16 E6 and E7 Fusion Gene in Vivo and in Vitro

Background: : Cervical cancer is the second most common cause of cancer related death of women. PersistentHPV infection, especially with high-risk types such as HPV16 and HPV18, has been identified to be the primarycause of cervical cancer. E6 and E7 are the major oncoproteins of high-risk HPVs, which are expressed exclusivelyin HPV infected tissues, and thereby represent ideal therapeutic targets for immunotherapy of cervical cancer.Materials and Methods: In this work, we used recombinant adenovirus expressing coden-optimized HPV16 E6and E7 fusion protein (Ad-ofE6E7) to prime dendritic cells (DC-ofE6E7), to investigate the ability of primed DCvaccine in eliciting antitumor immunity in vitro and vivo. Results: Our results indicated that DC-ofE6E7 vaccineco-culturing with splenocytes could strongly induce a tumor-specific cytotoxic T lymphocyte (CTL) responseand kill the TC-1 cells effectively in vitro. Moreover, DC-ofE6E7 vaccine induced protective immunity againstthe challenge of TC-1 cancer cells in vivo. Conclusions: The results suggested that the HPV16 ofE6E7 primedDC vaccine has potential application for cervical cancer immunotherapy.     

Pre-clinical immunogenicity and anti-tumour efficacy of a deleted recombinant human papillomavirus type 16 E7 protein

BACKGROUND: Current vaccination strategies against Human papillomavirus (HPV)-induced ano-genital cancers mostly target E7 from HPV16. However, the oncogenic nature of E7 raises potential human safety issues. Although the modifications abrogating the E7 transforming potential have been well characterized, their effect on E7 immunogenicity has been poorly studied. In this study, we evaluated the vaccine potential of an HPV16 E7 protein deleted from the entire pRb-binding motif. MATERIALS AND METHODS: Purified recombinant deleted (E7delta21-26) and wild-type (His6-E7 and E7WT) E7 proteins were studied in pre-clinical mice models. RESULTS: In C57BL/6 mice, E7delta21-26 formulated with the Quil A adjuvant generated systemic E7-specific cytotoxic T-cell and antibody responses similar to those induced following His6-E7/Quil A and E7WT/Quil A vaccinations. E7delta21-26/Quil A injections efficiently protected animals from challenge with the HPV16-expressing tumours, C3 and TC-1. Moreover, therapeutic vaccination with adjuvant-modified E7 suppressed or significantly decreased C3 tumour outgrowth. CONCLUSION: E7delta21-26 could represent a safe and efficient vaccine candidate against E7-containing tumour cells.     

表达HPV16 E6和E7蛋白的非复制型重组痘苗病毒诱发的抗肿瘤免疫反应

目的　观察表达人乳头瘤病毒 (HPV) 16E6和E7蛋白的非复制型重组痘苗病毒的抗肿瘤免疫效果。方法　以重组痘苗病毒NTVJmE6E7免疫C5 7BL/ 6小鼠 ,检测特异性的细胞毒性T淋巴细胞 (CTL)活性 ;经免疫后的小鼠以TC 1肿瘤细胞攻击 ,观察免疫保护效果 ;荷瘤小鼠切除肿瘤后接种重组痘苗病毒 ,观察肿瘤复发情况。结果　以重组痘苗病毒NTVJmE6E7免疫小鼠 ,可诱导产生针对TC 1细胞的特异性的CTL反应 ;加强免疫后的小鼠能耐受 1× 10 4TC 1细胞的攻击 ;以重组痘苗病毒NTVJmE6E7免疫肿瘤术后小鼠 ,能有效地预防肿瘤复发。结论　非复制型重组痘苗病毒NTVJmE6E7可作为HPV16的相关肿瘤及其癌前病变免疫治疗的候选疫苗。     

Effect of adenovirus-mediated heat shock protein expression and oncolysis in combination with low-dose cyclophosphamide treatment on antitumor immune responses

Heat shock proteins such as gp96 have the ability to chaperone peptides and activate antigen-presenting cells. In this study, we tested whether adenovirus-mediated overexpression of secreted or membrane-associated forms of gp96 in tumor cells would stimulate an antitumor immune response. Studies were carried out in C57Bl/6 mice bearing aggressively growing s.c. tumors derived from syngeneic TC-1 cells, a cell line that expresses HPV16 E6 and E7 proteins. We found that secreted gp96 can induce protective and therapeutic antitumor immune responses. Our data also indicate that the antitumor effect of sgp96 expression seems to be limited by the induction of suppressive regulatory T cells (Treg). TC-1 tumor transplantation increased the number of splenic and tumor-infiltrating Tregs. Importantly, treatment of mice with low-dose cyclophosphamide decreased the number of Tregs and enhanced the immunostimulatory effect of sgp96 expression. We also tested whether an oncolytic vector (Ad.IR-E1A/TRAIL), that is able to induce tumor cell apoptosis and, potentially, release cryptic tumor epitopes in immunogenic form, could stimulate antitumor immune responses. Although tumor cells infected ex vivo with Ad.IR-E1A/TRAIL had no antitumor effect when used as a vaccine alone, the additional treatment with low-dose cyclophosphamide resulted in the elimination of pre-established tumors. This study gives a rationale for testing approaches that suppress Tregs in combination with oncolytic or immunostimulatory vectors.     

Eradication of established tumors by vaccination with recombinant Bordetella pertussis adenylate cyclase carrying the human papillomavirus 16 E7 oncoprotein

High-risk human papillomaviruses (HPV) such as HPV16 are associated with the development of cervical cancer. The HPV16-E6 and HPV16-E7 oncoproteins are expressed throughout the replicative cycle of the virus and are necessary for the onset and maintenance of malignant transformation. Both these tumor-specific antigens are considered as potential targets for specific CTL-mediated immunotherapy. The adenylate cyclase (CyaA) of Bordetella pertussis is able to target dendritic cells through specific interaction with the alpha(M)beta(2) integrin. It has been previously shown that this bacterial protein could be used to deliver CD4(+) and CD8(+) T cell epitopes to the MHC class II and class I presentation pathways to trigger specific Th and CTL responses in vivo, providing protection against subsequent viral or tumoral challenge. Here, we constructed recombinant CyaA containing either the full sequence or various subfragments from the HPV16-E7 protein. We show that, when injected to C57BL/6 mice in absence of any adjuvant, these HPV16-recombinant CyaAs are able to induce specific Th1 and CTL responses. Furthermore, when injected into mice grafted with HPV16-E7-expressing tumor cells (TC-1), one of these recombinant proteins was able to trigger complete tumor regression in 100% of the animals tested. This therapeutic efficacy compared favorably to that of strongly adjuvanted peptide and was marginally affected by prior immunity to CyaA protein. This study represents the first in vivo demonstration of the antitumoral therapeutic activity of recombinant CyaA proteins carrying human tumor-associated antigens and paves the way for the testing of this vector in clinical trials.     

Human papillomavirus type 16 E6 and E7 genotypes in head-and-neck carcinomas

Human papillomavirus type 16 (HPV16) is associated with squamous cell carcinomas of the head and neck (HNSCC) particularly from the Waldeyer's tonsillar ring. A causal role of HPV16 in carcinogenesis is linked to the activity of the viral oncoproteins E6 and E7 which inactivate the cellular tumor suppressors p53 and pRB, respectively. Lack of E6 expression in HPV16-positive HNSCC has been reported, in some cases caused by disruption of the E6 gene. We have examined the status of the HPV16 E6-E7 gene region in tumor and metastasis samples of 24 HNSCC patients employing genomic PCR. No cases with a disrupted E6-E7 region could be identified. Sequence analysis of the E6-E7 segments revealed three different HPV16 E6-E7 genotypes: the HPV16 prototype (6 of 21 cases), the E6 variant T350G (8 of 21 cases), and the E6-E7 variant A131G/C712A (7 of 21 cases). The E6 variants T350G and A131G have been associated with increased oncogenic potential in cervical cancer patients depending on host genetic factors. Their high prevalence in the HNSCC samples studied indicates that they may be important also in HNSCC development.     

Photodynamic therapy-generated tumor cell lysates with CpG-oligodeoxynucleotide enhance immunotherapy efficacy in human papillomavirus 16 (E6/E7) immortalized tumor cells

Immunotherapy with photodynamic therapy (PDT) offers great promise as a new alternative for cancer treatment; however, its use remains experimental. In this study, we examined the immunotherapeutic significance of human papillomavirus (HPV)-immortalized tumor cell lysates induced by PDT with CpG-oligodeoxynucleotide (ODN). PDT-cell lysates were generated by irradiating Radachlorin (5 microg/mL) preloaded TC-1 cells carrying HPV 16 E7. PDT-cell lysates plus ODN coinjection for protection against E7-expressing tumors as well as specific immune responses were evaluated with the following tests: heat shock protein 70 (HSP70) enzyme-linked immunosorbent assay, in vitro and in vivo tumor growth inhibition, interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) assay, cytotoxic T-lymphocyte assay, and fluorescence activated cell sorting (FACS) analysis. PDT-cell lysates plus ODN coinjection showed a significant suppression of tumor growth at both prophylactic and therapeutic levels, compared to PDT (or F/T)-cell lysates or ODN alone. In addition, we evaluated the level of the immune response with the coinjection. HSP70, an important regulator of inflammatory and immune response, was observed in abundance in the PDT-cell lysates. IFN-gamma production and cytotoxic T lymphocytes (CTL) responses were induced by PDT-cell lysates plus ODN injection. The coinjection resulted in PDT-cell lysate-specific antibodies (IgG1, IgG2a, IgG2b, and IgG3) and T-helper cell responses significantly higher than PDT-cell lysates alone. Moreover, IFN-gamma production and CTL responses were significantly induced in the PDT-cell lysate plus ODN immunized groups. These enhanced immune responses appeared to be mediated by CD8+ T cells only. These data suggest that PDT-cell lysates plus ODN injection may be an effective approach to induce CTL immune responses as a possible immunotherapeutic strategy for cancer therapy.