Toll样受体激动剂和RNA干预技术对HPV 6b/11型感染的抑制作用研究
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摘要
尖锐湿疣(Condyloma acuminatum,CA)是我国发病率最高的性传播疾病之一,临床上顽固难治,极易复发,严重危害患者的身心健康,寻求理想的治疗和预防手段迫在眉睫。人乳头瘤病毒(Human papillomavirus,HPV)是尖锐湿疣的病原体,其中6型和11型是CA的主要致病型别,相对于肿瘤相关的高危型HPV,主要感染皮肤和外生殖器粘膜,被称为低危型或皮肤型HPV。HPV感染常不能有效激发机体免疫应答以清除病毒感染,是HPV感染难治的主要原因。如何有效抑制病毒复制,激发机体特异性抗HPV免疫应答是控制或清除HPV感染的关键。HPV早期基因E7是主要致病基因之一,可引起宿主细胞增殖周期改变,并下调抑癌基因表达,诱导细胞恶性转化,因而是HPV感染防治研究的理想靶点。本研究以E7基因或蛋白为研究基础,从调节宿主抗病毒特异性细胞免疫、和特异性沉默主要致病基因两个方面进行HPV感染的干预实验,以期为CA等低危型HPV感染相关疾病的防治研究提供一定的实验依据。
Toll样受体(Toll like receptors,TLRs)能特异性识别病原微生物进化过程中保守的病原相关分子模式(pathogen-associated molecular patterns,PAMPs),启动天然免疫应答并继而激活获得性免疫反应。通过激活某些TLR信号通路,可促进Th0细胞(Th,T helper,辅助性T细胞)向Th1分化,调节T细胞亚群Th1/Th2、Tc1/Tc2的平衡,促进抗原特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL,Tc)的活性,有利于病毒感染细胞的清除。本研究以人外周血单核细胞来源的树突状细胞(monocyte-derived dendritic cells,mdDCs)负载HPV 11 E7抗原HLA-A~*0201限制性CTL优势表位肽(以下简称E7多肽),以不同TLR激动剂诱导其成熟,分析其对mdDCs的表型、细胞因子分泌水平及其对T淋巴细胞的活化作用的影响。结果显示,所研究的TLR激动剂均能促进mdDCs功能分化及成熟,促进负载E7多肽的mdDCs(以下简称E7-mdDCs)分泌IL-12,尤以TLR3的激动剂聚肌胞苷酸(polyinosinicacid-polycytidylic acid,PIC)和TLR4的激动剂脂多糖(lipopolysaccharide,LPS)刺激作用最强,而TLR7的激动剂咪喹莫特及TLR9的激动剂胞嘧啶鸟嘌呤核苷酸序列(cytidylyl phosphate guanosine oligoneuleotid,CpG ODN)的作用相对较弱。PIC诱导的E7-mdDCs能促使CD4~+初始T细胞分泌高水平IFN-γ,作用明显强于LPS、咪喹莫特及CpG ODN组。此外,LPS和PIC诱导的E7-mdDCs可提高T淋巴细胞分泌IFN-γ、IL-2、TNF-α的水平以及分泌IFN-γ、IL-2的T淋巴细胞频数,并显著增强E7特异性CTL活性,作用明显高于另两个激动剂咪喹莫特和CpG ODN。结果提示TLR激动剂可上调HPV11 E7 CTL表位肽诱导的特异性免疫应答,进而有利于控制或清除HPV 11型感染细胞。而在进一步基于mdDCs的多肽疫苗研究中,TLR3和TLR4的激动剂可能成为有效的疫苗佐剂。
特异性HPV疫苗在预防HPV感染方面显示了高保护率,但对于免疫抑制的患者仍效果不佳,因为它们在这类患者体内仍无法激发有效的免疫保护应答,因而可能需从清除病毒本身的角度来进行干预。RNA干扰(RNA interference,RNAi)指双链RNA在生物细胞内对序列特异性mRNA表达的抑制作用,具特异性和高效性,因而作为一种重要的基因沉默技术被广泛应用于基因功能研究及疾病机制和防治研究中。本文在表达HPV6b或11型E7基因的小鼠黑素瘤细胞株BL6-B16和小鼠荷瘤模型中研究siRNA(small interfering RNA)和shRNA(small hairpin RNA)质粒表达载体对HPV基因早期基因E6、E7表达的沉默作用。特异性siRNA二聚体或shRNA质粒表达载体分别转染靶细胞HPV6bE7/B16和HPV11E7/B16,分析其在转染后不同时间、及不同转染剂量下对细胞内靶基因mRNA表达的影响。表达HPV6b或11型E7基因的荷瘤小鼠模型则以瘤内注射或尾静脉注射法导入阳离子脂质体负载的siRNA或shRNA质粒表达载体,注射3次后检测肿瘤组织内靶基因mRNA的表达。检测方法为实时荧光定量PCR。体外实验结果表明,siRNA及shRNA质粒表达载体均可在体外高效特异地抑制HPV6b和11型E7基因的表达,且其干预作用存在一定的量效性和时效性。SiRNA- HPV6b/11 E7的最佳作用浓度为25-50 nmol/L,shRNA质粒表达载体pU6-sh6b/11 E7的最佳作用浓度为0.2-0.4μg/ml,靶基因表达抑制率均于72h达到最高(60%-80%),抑制效果可持续96h。动物实验结果表明,siRNA及shRNA质粒表达载体分别以瘤内注射或尾静脉注射法导入小鼠模型,可明显抑制肿瘤内E7基因的表达,且瘤内注射较尾静脉注射效果更明显,瘤内注射对靶基因表达的抑制效率可达50%以上。结果提示siRNA及shRNA质粒表达载体在体外培养细胞及小鼠体内均可有效特异地抑制HPV6b/11 E7基因的表达,提示RNAi技术可通过对HPV6b/11 E7基因的特异性沉默干预HPV6b/11病毒蛋白的合成、抑制蛋白功能进而影响病毒复制乃至病毒播散。该技术将有利于CA等低危型HPV感染疾病的控制。
结论:本研究不仅提示TLR激动剂(尤其是TLR3的激动剂PIC和TLR4的激动剂LPS)可能作为有效的免疫佐剂应用于HPV多肽-DCs疫苗研究,以有效控制或清除HPV感染细胞,还提示两种RNAi策略在体外培养细胞和动物模型中诱导的特异性6b/11型E7基因沉默可能通过抑制HPV病毒的复制而有利于控制疾病发展和传播,从免疫学治疗策略及基因治疗策略两个方面为低危型HPV感染相关性疾病尤其是CA的防治研究提供了一定的实验依据。
Condyloma acuminatum(CA) is one of the most common sexually transmitted diseases. The complete cure is difficult to achieve because of its high recurrence.CA-related emotional distress and economic burdens have been noticed,thus there is a serious need for effective strategies to prevent or treat CA.Human papilloma virus(HPV) infection is the cause of CA.The types 6 and 11,as compared to the carcinoma-related "high-risk HPVs",are "low-risk HPVs" which are usually involved in skin and mucosal genitalia, so as to be responsible for most of CA.Since the insufficient elicitation of immune protection due to the impaired systemic or local immunity in persistent HPV infections, it is difficult to elimimate HPV-infected cells efficiently.Thus,how to inhibit the viral replication and how to raise the efficient immune responses,especially the specific cytotoxic T lymphocyte(CTL) activity,against HPVs by host remains a research emphasis.Early gene E7 of HPVs plays a crucial role in cell proliferation,induction and maintenance of malignant transformation of HPV-associated lesions.Therefore,the E7 gene or antigen represents an ideal target for development of therapies against HPVs. The current research based on E7 gene or antigen is focused on the modulation of specific anti-viral cellular immunity,as well as the specific silence of E7 gene expression.It may provide laboratory evidences on the prophylaxis and treatment of low-risk HPV infection-related diseases such as CA.
Toll like receptors(TLRs) recognize pathogen-associated molecular patterns (PAMPs ) for early recognition of microbial invasion and thus play a key role in innate immunity and subsequently in adaptive immunity.The activation of several TLR signals could benefit the ThO cells differentiating into Th1 cells,so as to repair the Th1/Th2 or Tc1/Tc2 imbalance,and to promote pathogen-specific CTL responses.Thus TLR activation may benifit the elimination of HPV-infected cells.To analyze the substantial effects of TLR agonists as adjuvants of a HPV protein vaccine regimen,we studied the ability of various TLR agonists to modulate the maturation and the cytokine production of HPV 11 E7 protein HLA-A*0201 stricted CTL epitope(E7 peptide) -loaded monocyte-derived dendritic cells(mdDCs),and the ability of mdDCs to further enhance Th1 responses and E7-specific CTL activity.The results showed all the studied TLR agonists,especially TLR3 agonist(polyinosinic acid-polycytidylic acid,PIC) and TLR4 agonist(lipopolysaccharide,LPS),could promote the maturation and IL-12 production of E7-loaded mdDCs(E7-mdDCs).The effect of TLR7 agonist(imiquimod) and TLR9 agonist(cytidylyl phosphate guanosine oligoneuleotid,CpG ODN) were relatively weak.PIC-induced E7-mdDCs were much more effective than the other three agonists in enhancing the IFN-γsecretion of CD4~+ naive cells.Additionally,the E7-mdDCs stimulated with LPS or PIC increased the level of IFN-y,IL-2,and TNF-αsecreted by effector T cells,and increased the frequencies of the IFN-γ- or the IL-2-secreting T cells,as well as the E7-specific CTL activity.However,as compared with LPS and PIC,the effects of imiquimod and CpG ODN were relatively poor.These data indicated that TLR agonists might favour the elimination of HPV-infected cells within CA lesions through the enhanced immune responses induced by E7 peptide-vaccine.Both the agonists of TLR3 and TLR4 might be potent adjuvants in the further research of mdDCs-based vaccine regimens against CA.
RNA interference(RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of messenger RNA in animal and plant cells.The process is highly efficient and specific.RNAi is now established as an important biological strategy for gene silencing.It has been applied in dissection of gene function, mechanism and therapeutic research of various diseases.Here we introduced RNAi into HPV research and asked if small interfering RNA(siRNA) duplex and small hairpin RNA(shRNA)-expressing vector can be employed to silence exogenous HPV gene expression in mouse melanoma cells BL6-B16 or in mouse model expressing HPV 6b or 11 E7 gene.Cells were transfected with siRNA or siRNA-expressing vector and harvested at different time points,or be transfected with various doses of siRNA or siRNA-expressing vector.The target gene expression was analyzed with Real-time PCR. The mice were received three repeated intratumoral or intravenous injection of siRNAs or siRNA-expressing vectors with cationic liposomes,and the E7 gene expression was also analyzed.The in-vitro results showed that both siRNA and siRNA-expressing vector significantly reduced the mRNA expression of HPV6b E7 and HPV 11E7 in a moderate dose-dependent and time-dependent manner.SiRNAs and shRNA-expressing vector reduced the mRNA levels of HPV 6b E7 or HPV 11 E7 to 60%- 80%with an optimal dosage of 25-50 nmol/L(siRNAs) and 0.2μg/ml -0.4μg/ml(shRNA-expressing vectors),respectively.The optimal time is 72 h,and the inhibitory effect sustained for at least 96h.The in-vivo experiment showed that the delivery of siRNA or siRNA-expressing vector in mice induced a moderate inhibitory effect of targeted gene expression in tumor tissues.The intratumoral injection was more effective as compared to the intravenous injection.And E7 mRNA expression in tumor models was reduced up to 50%after three I.T.injections.Our findings indicated that both RNAi approaches could specifically silence the gene expression of HPV6b/11 E7 in vitro and in vivo. RNAi may present another potential vehicle to handle HPV6b/11-related diseases such as CA,through the functional interference of E7 protein and the inhibition of the viral replication.
Conclusion:Taken together,the project not only indicated that TLRs agonists might be applied as adjuvants in further research on peptide-DCs vaccine regimens to eliminate HPV-infected cells,but also suggested that the potent specific inhibition of E7 gene expression might benefit the inhibition of HPV viral replication the suppression of CA progress and dissenmination.This project provided evidences on both immunotherapy strateigies and gene-therapy strateigies for CA management.It will be helpful to the development of new approaches for low-risk HPV infectious diseases, especially for CA.
Toll样受体(Toll like receptors,TLRs)能特异性识别病原微生物进化过程中保守的病原相关分子模式(pathogen-associated molecular patterns,PAMPs),启动天然免疫应答并继而激活获得性免疫反应。通过激活某些TLR信号通路,可促进Th0细胞(Th,T helper,辅助性T细胞)向Th1分化,调节T细胞亚群Th1/Th2、Tc1/Tc2的平衡,促进抗原特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL,Tc)的活性,有利于病毒感染细胞的清除。本研究以人外周血单核细胞来源的树突状细胞(monocyte-derived dendritic cells,mdDCs)负载HPV 11 E7抗原HLA-A~*0201限制性CTL优势表位肽(以下简称E7多肽),以不同TLR激动剂诱导其成熟,分析其对mdDCs的表型、细胞因子分泌水平及其对T淋巴细胞的活化作用的影响。结果显示,所研究的TLR激动剂均能促进mdDCs功能分化及成熟,促进负载E7多肽的mdDCs(以下简称E7-mdDCs)分泌IL-12,尤以TLR3的激动剂聚肌胞苷酸(polyinosinicacid-polycytidylic acid,PIC)和TLR4的激动剂脂多糖(lipopolysaccharide,LPS)刺激作用最强,而TLR7的激动剂咪喹莫特及TLR9的激动剂胞嘧啶鸟嘌呤核苷酸序列(cytidylyl phosphate guanosine oligoneuleotid,CpG ODN)的作用相对较弱。PIC诱导的E7-mdDCs能促使CD4~+初始T细胞分泌高水平IFN-γ,作用明显强于LPS、咪喹莫特及CpG ODN组。此外,LPS和PIC诱导的E7-mdDCs可提高T淋巴细胞分泌IFN-γ、IL-2、TNF-α的水平以及分泌IFN-γ、IL-2的T淋巴细胞频数,并显著增强E7特异性CTL活性,作用明显高于另两个激动剂咪喹莫特和CpG ODN。结果提示TLR激动剂可上调HPV11 E7 CTL表位肽诱导的特异性免疫应答,进而有利于控制或清除HPV 11型感染细胞。而在进一步基于mdDCs的多肽疫苗研究中,TLR3和TLR4的激动剂可能成为有效的疫苗佐剂。
特异性HPV疫苗在预防HPV感染方面显示了高保护率,但对于免疫抑制的患者仍效果不佳,因为它们在这类患者体内仍无法激发有效的免疫保护应答,因而可能需从清除病毒本身的角度来进行干预。RNA干扰(RNA interference,RNAi)指双链RNA在生物细胞内对序列特异性mRNA表达的抑制作用,具特异性和高效性,因而作为一种重要的基因沉默技术被广泛应用于基因功能研究及疾病机制和防治研究中。本文在表达HPV6b或11型E7基因的小鼠黑素瘤细胞株BL6-B16和小鼠荷瘤模型中研究siRNA(small interfering RNA)和shRNA(small hairpin RNA)质粒表达载体对HPV基因早期基因E6、E7表达的沉默作用。特异性siRNA二聚体或shRNA质粒表达载体分别转染靶细胞HPV6bE7/B16和HPV11E7/B16,分析其在转染后不同时间、及不同转染剂量下对细胞内靶基因mRNA表达的影响。表达HPV6b或11型E7基因的荷瘤小鼠模型则以瘤内注射或尾静脉注射法导入阳离子脂质体负载的siRNA或shRNA质粒表达载体,注射3次后检测肿瘤组织内靶基因mRNA的表达。检测方法为实时荧光定量PCR。体外实验结果表明,siRNA及shRNA质粒表达载体均可在体外高效特异地抑制HPV6b和11型E7基因的表达,且其干预作用存在一定的量效性和时效性。SiRNA- HPV6b/11 E7的最佳作用浓度为25-50 nmol/L,shRNA质粒表达载体pU6-sh6b/11 E7的最佳作用浓度为0.2-0.4μg/ml,靶基因表达抑制率均于72h达到最高(60%-80%),抑制效果可持续96h。动物实验结果表明,siRNA及shRNA质粒表达载体分别以瘤内注射或尾静脉注射法导入小鼠模型,可明显抑制肿瘤内E7基因的表达,且瘤内注射较尾静脉注射效果更明显,瘤内注射对靶基因表达的抑制效率可达50%以上。结果提示siRNA及shRNA质粒表达载体在体外培养细胞及小鼠体内均可有效特异地抑制HPV6b/11 E7基因的表达,提示RNAi技术可通过对HPV6b/11 E7基因的特异性沉默干预HPV6b/11病毒蛋白的合成、抑制蛋白功能进而影响病毒复制乃至病毒播散。该技术将有利于CA等低危型HPV感染疾病的控制。
结论:本研究不仅提示TLR激动剂(尤其是TLR3的激动剂PIC和TLR4的激动剂LPS)可能作为有效的免疫佐剂应用于HPV多肽-DCs疫苗研究,以有效控制或清除HPV感染细胞,还提示两种RNAi策略在体外培养细胞和动物模型中诱导的特异性6b/11型E7基因沉默可能通过抑制HPV病毒的复制而有利于控制疾病发展和传播,从免疫学治疗策略及基因治疗策略两个方面为低危型HPV感染相关性疾病尤其是CA的防治研究提供了一定的实验依据。
Condyloma acuminatum(CA) is one of the most common sexually transmitted diseases. The complete cure is difficult to achieve because of its high recurrence.CA-related emotional distress and economic burdens have been noticed,thus there is a serious need for effective strategies to prevent or treat CA.Human papilloma virus(HPV) infection is the cause of CA.The types 6 and 11,as compared to the carcinoma-related "high-risk HPVs",are "low-risk HPVs" which are usually involved in skin and mucosal genitalia, so as to be responsible for most of CA.Since the insufficient elicitation of immune protection due to the impaired systemic or local immunity in persistent HPV infections, it is difficult to elimimate HPV-infected cells efficiently.Thus,how to inhibit the viral replication and how to raise the efficient immune responses,especially the specific cytotoxic T lymphocyte(CTL) activity,against HPVs by host remains a research emphasis.Early gene E7 of HPVs plays a crucial role in cell proliferation,induction and maintenance of malignant transformation of HPV-associated lesions.Therefore,the E7 gene or antigen represents an ideal target for development of therapies against HPVs. The current research based on E7 gene or antigen is focused on the modulation of specific anti-viral cellular immunity,as well as the specific silence of E7 gene expression.It may provide laboratory evidences on the prophylaxis and treatment of low-risk HPV infection-related diseases such as CA.
Toll like receptors(TLRs) recognize pathogen-associated molecular patterns (PAMPs ) for early recognition of microbial invasion and thus play a key role in innate immunity and subsequently in adaptive immunity.The activation of several TLR signals could benefit the ThO cells differentiating into Th1 cells,so as to repair the Th1/Th2 or Tc1/Tc2 imbalance,and to promote pathogen-specific CTL responses.Thus TLR activation may benifit the elimination of HPV-infected cells.To analyze the substantial effects of TLR agonists as adjuvants of a HPV protein vaccine regimen,we studied the ability of various TLR agonists to modulate the maturation and the cytokine production of HPV 11 E7 protein HLA-A*0201 stricted CTL epitope(E7 peptide) -loaded monocyte-derived dendritic cells(mdDCs),and the ability of mdDCs to further enhance Th1 responses and E7-specific CTL activity.The results showed all the studied TLR agonists,especially TLR3 agonist(polyinosinic acid-polycytidylic acid,PIC) and TLR4 agonist(lipopolysaccharide,LPS),could promote the maturation and IL-12 production of E7-loaded mdDCs(E7-mdDCs).The effect of TLR7 agonist(imiquimod) and TLR9 agonist(cytidylyl phosphate guanosine oligoneuleotid,CpG ODN) were relatively weak.PIC-induced E7-mdDCs were much more effective than the other three agonists in enhancing the IFN-γsecretion of CD4~+ naive cells.Additionally,the E7-mdDCs stimulated with LPS or PIC increased the level of IFN-y,IL-2,and TNF-αsecreted by effector T cells,and increased the frequencies of the IFN-γ- or the IL-2-secreting T cells,as well as the E7-specific CTL activity.However,as compared with LPS and PIC,the effects of imiquimod and CpG ODN were relatively poor.These data indicated that TLR agonists might favour the elimination of HPV-infected cells within CA lesions through the enhanced immune responses induced by E7 peptide-vaccine.Both the agonists of TLR3 and TLR4 might be potent adjuvants in the further research of mdDCs-based vaccine regimens against CA.
RNA interference(RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of messenger RNA in animal and plant cells.The process is highly efficient and specific.RNAi is now established as an important biological strategy for gene silencing.It has been applied in dissection of gene function, mechanism and therapeutic research of various diseases.Here we introduced RNAi into HPV research and asked if small interfering RNA(siRNA) duplex and small hairpin RNA(shRNA)-expressing vector can be employed to silence exogenous HPV gene expression in mouse melanoma cells BL6-B16 or in mouse model expressing HPV 6b or 11 E7 gene.Cells were transfected with siRNA or siRNA-expressing vector and harvested at different time points,or be transfected with various doses of siRNA or siRNA-expressing vector.The target gene expression was analyzed with Real-time PCR. The mice were received three repeated intratumoral or intravenous injection of siRNAs or siRNA-expressing vectors with cationic liposomes,and the E7 gene expression was also analyzed.The in-vitro results showed that both siRNA and siRNA-expressing vector significantly reduced the mRNA expression of HPV6b E7 and HPV 11E7 in a moderate dose-dependent and time-dependent manner.SiRNAs and shRNA-expressing vector reduced the mRNA levels of HPV 6b E7 or HPV 11 E7 to 60%- 80%with an optimal dosage of 25-50 nmol/L(siRNAs) and 0.2μg/ml -0.4μg/ml(shRNA-expressing vectors),respectively.The optimal time is 72 h,and the inhibitory effect sustained for at least 96h.The in-vivo experiment showed that the delivery of siRNA or siRNA-expressing vector in mice induced a moderate inhibitory effect of targeted gene expression in tumor tissues.The intratumoral injection was more effective as compared to the intravenous injection.And E7 mRNA expression in tumor models was reduced up to 50%after three I.T.injections.Our findings indicated that both RNAi approaches could specifically silence the gene expression of HPV6b/11 E7 in vitro and in vivo. RNAi may present another potential vehicle to handle HPV6b/11-related diseases such as CA,through the functional interference of E7 protein and the inhibition of the viral replication.
Conclusion:Taken together,the project not only indicated that TLRs agonists might be applied as adjuvants in further research on peptide-DCs vaccine regimens to eliminate HPV-infected cells,but also suggested that the potent specific inhibition of E7 gene expression might benefit the inhibition of HPV viral replication the suppression of CA progress and dissenmination.This project provided evidences on both immunotherapy strateigies and gene-therapy strateigies for CA management.It will be helpful to the development of new approaches for low-risk HPV infectious diseases, especially for CA.
引文
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