The Inhibitory Effect Of Toll Like Receptors Angonists And RNA Interference On HPV Type 6b/11 Infection

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.