A DNA Vaccine Encoding a Codon-Optimized Human Papillomavirus Type 16 E6 Gene Enhances CTL Response and Anti-tumor Activity

Summary The HPV oncoproteins E6 and E7 are consistently expressed in HPV-associated cancer cells and are responsible for their malignant transformation. Therefore, HPV E6 and E7 are ideal target antigens for developing vaccines and immunotherapeutic strategies against HPV-associated neoplasms. Recently, it has been demonstrated that codon optimization of the HPV-16 E7 gene resulted in highly efficient translation of E7 and increased the immunogenicity of E7-specific DNA vaccines. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a codon-optimized HPV-16 E6 DNA vaccine (pNGVL4a-E6/opt) and characterized the E6-specific CD8⁺ T cell immune responses as well as the protective and therapeutic anti-tumor effects in vaccinated C57BL/6 mice. Our data indicated that transfection of human embryonic kidney cells (293 cells) with pNGVL4a-E6/opt resulted in highly efficient translation of E6. In addition, vaccination with pNGVL4a-E6/opt significantly enhanced E6-specific CD8⁺ T cell immune responses in C57BL/6 mice. Mice vaccinated with pNGVL4a-E6/opt are able to generate potent protective and therapeutic antitumor effects against challenge with E6-expressing tumor cell line, TC-1. Thus, DNA vaccines encoding a codon-optimized HPV-16 E6 may be a promising strategy for improving the potency of prophylactic and therapeutic HPV vaccines with potential clinical implications.     

Design,Immune Responses and Anti-Tumor Potential of an HPV16 E6E7 Multi-Epitope Vaccine

Cervical cancer is a common type of cancer among women worldwide and infection with high-risk human papillomavirus (HPVs) types represents the major risk factor for the etiopathogenesis of the disease. HPV-16 is the most frequently identified HPV type in cervical lesions and expression of E6 and E7 oncoproteins is required for the uncontrolled cellular proliferation. In the present study we report the design and experimental testing of a recombinant multi-epitope protein containing immunogenic epitopes of HPV-16 E6 and E7. Tumor preventive assays, based on the engraftment of TC-1 cells in mice, showed that the E6E7 multi-epitope protein induced a full preventive anti-tumor protection in wild-type mice, as well as in mice deficient in expression of CD4⁺ T cells and TLR4 receptor. Nonetheless, no anti-tumor protection was observed in mice deficient in CD8⁺ T cells. Also, the vaccine promoted high activation of E6/E7-specific T cells and in a therapeutic-approach, E6E7 protein conferred full anti-tumor protection in mice. These results show a potential use of this E6E7 multi-epitope antigen as a new and promising antigen for the development of a therapeutic vaccine against tumors induced by HPV.     

Attenuated Recombinant Influenza A Virus Expressing HPV16 E6 and E7 as a Novel Therapeutic Vaccine Approach

Persistent infection with high-risk human papillomavirus (HPV) types, most often HPV16 and HPV18, causes all cervical and most anal cancers, and a subset of vulvar, vaginal, penile and oropharyngeal carcinomas. Two prophylactic virus-like particle (VLPs)-based vaccines, are available that protect against vaccine type-associated persistent infection and associated disease, yet have no therapeutic effect on existing lesions or infections. We have generated recombinant live-attenuated influenza A viruses expressing the HPV16 oncogenes E6 and E7 as experimental immunotherapeutic vaccine candidates. The influenza A virus life cycle lacks DNA intermediates as important safety feature. Different serotypes were generated to ensure efficient prime and boost immunizations. The immune response to vaccination in C57BL/6 mice was characterized by peptide ELISA and IFN-γ ELISpot, demonstrating induction of cell-mediated immunity to HPV16 E6 and E7 oncoproteins. Prophylactic and therapeutic vaccine efficacy was analyzed in the murine HPV16-positive TC-1 tumor challenge model. Subcutaneous (s.c.) prime and boost vaccinations of mice with recombinant influenza A serotypes H1N1 and H3N2, followed by challenge with TC-1 cells resulted in complete protection or significantly reduced tumor growth as compared to control animals. In a therapeutic setting, s.c. vaccination of mice with established TC-1 tumors decelerated tumor growth and significantly prolonged survival. Importantly, intralesional vaccine administration induced complete tumor regression in 25% of animals, and significantly reduced tumor growth in 50% of mice. These results suggest recombinant E6E7 influenza viruses as a promising new approach for the development of a therapeutic vaccine against HPV-induced disease.     

Design of a Highly Effective Therapeutic HPV16 E6/E7-Specific DNA Vaccine: Optimization by Different Ways of Sequence Rearrangements (Shuffling)

Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16) is the causative event for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. Despite many attempts to develop therapeutic vaccines no candidate has entered late clinical trials. An interesting approach is a DNA based vaccine encompassing the nucleotide sequence of the E6 and E7 viral oncoproteins. Because both proteins are consistently expressed in HPV infected cells they represent excellent targets for immune therapy. Here we report the development of 8 DNA vaccine candidates consisting of differently rearranged HPV-16 E6 and E7 sequences within one molecule providing all naturally occurring epitopes but supposedly lacking transforming activity. The HPV sequences were fused to the J-domain and the SV40 enhancer in order to increase immune responses. We demonstrate that one out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific cellular immune responses in mice and, as shown in regression experiments, efficiently controls growth of HPV 16 positive syngeneic tumors. This data demonstrates the potential of this vaccine candidate to control persistent HPV 16 infection that may lead to malignant disease. It also suggests that different sequence rearrangements influence the immunogenecity by an as yet unknown mechanism.     

Combined Stimulation of IL-2 and 4-1BB Receptors Augments the Antitumor Activity of E7 DNA Vaccines by Increasing Ag-Specific CTL Responses

Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer.     

Development of a DNA vaccine targeting human papillomavirus type 16 oncoprotein E6

Human papillomavirus (HPV), particularly type 16 (HPV-16), is present in more than 99% of cervical cancers. The HPV oncoproteins E6 and E7 are constantly expressed and therefore represent ideal targets for HPV vaccine development. We previously developed DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 and generated potent E7-specific CD8(+) T-cell immune responses and antitumor effects against an E7-expressing tumor. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a DNA vaccine encoding CRT linked to E6 (CRT/E6). Our results indicated that the CRT/E6 DNA vaccine, but not a wild-type E6 DNA vaccine, generated significant E6-specific CD8(+) T-cell immune responses in vaccinated mice. Mapping of the immunodominant epitope of E6 revealed that an E6 peptide comprising amino acids (aa) 48 to 57 (E6 aa48-57), presented by H-2K(b), is the optimal peptide and that the region of E6 comprising aa 50 to 57 represents the minimal core sequence required for activating E6-specific CD8(+) T lymphocytes. We also demonstrated that E6 aa48-57 contains cytotoxic T-lymphocyte epitopes naturally presented by E6-expressing TC-1 cells. Vaccination with a CRT/E6 but not a CRT/mtE6 (lacking aa 50 to 57 of E6) DNA vaccine could protect vaccinated mice from challenge with E6-expressing TC-1 tumors. Thus, our data indicate that E6 aa48-57 contains the immunodominant epitope and that a CRT/E6 DNA vaccine may be useful for control of HPV infection and HPV-associated lesions.     

人乳头瘤病毒16型E7蛋白的原核表达与鉴定

本研究在大肠杆菌BL21中融合表达人乳头瘤病毒16型E7蛋白,并初步评价其应用价值。采用PCR技术扩增出HPV16E7基因,将其克隆进原核表达载体pGEX6p-1,转化至大肠杆菌BL21,利用IPTG进行诱导表达。以纯化的融合蛋白作为检测抗原建立间接ELISA方法,用于检测重组李斯特菌(Lm1-2-E7)免疫小鼠后的E7血清抗体水平。在25℃,0.5mM IPTG诱导下,HPV16E7蛋白在大肠杆菌BL21中获得表达,融合蛋白以可溶性形式存在,Western blot结果显示其与HPV16E7单克隆抗体发生特异性反应。二次免疫后小鼠血清经间接ELISA结果表明E7特异性抗体滴度为1∶200。结果表明GST-E7融合蛋白具有较强的免疫活性。     

Characterization of HPV-16 E6 DNA vaccines employing intracellular targeting and intercellular spreading strategies

Summary Human papillomavirus (HPV) E6 and E7 are consistently expressed and are responsible for the malignant transformation of HPV-associated lesions. Thus, E6 and E7 represent ideal targets for therapeutic HPV vaccine development. We have previously used the gene gun approach to test several intracellular targeting and intercellular spreading strategies targeting HPV-16 E7. These strategies include the use of the sorting signal of lysosome-associated membrane protein (LAMP-1), Mycobacterium tuberculosis heat shock protein 70 (HSP70), calreticulin (CRT) and herpes simplex virus type 1 (HSV-1) VP22 proteins. All of these strategies have been shown to be capable of enhancing E7-DNA vaccine potency. In the current study, we have characterized DNA vaccines employing these intracellular targeting or intercellular spreading strategies targeting HPV-16 E6 for their ability to generate E6-specific CD8⁺ T cell immune responses and antitumor effects against an E6-expressing tumor cell line, TC-1, in C57BL/6 mice. We found that all the intracellular targeting strategies (CRT, LAMP-1, HSP70) as well as the intercellular spreading strategy (VP22) were able to enhance E6 DNA vaccine potency, although the orientation of HSP70 linked to E6 antigen in the E6 DNA vaccine appears to be important for the HSP70 strategy to work. The enhanced E6-specific CD8⁺ T cell immune response in vaccinated mice also translated into potent antitumor effects against TC-1 tumor cells. Our data indicate that all of the intracellular targeting and intercellular spreading strategies that have been shown to enhance E7 DNA vaccine potency were also able to enhance E6 DNA vaccine potency.     

重叠PCR合成HPV16E6、E7基因并构建植物表达双元载体

HPV16型为主的多种HPV病毒可诱发机体发生宫颈癌等疾病,以重叠PCR法人工合成HPV16 E6、E7致癌基因的融合基因,以之为目的基因构建了无选择标记基因(Marker-Free)双元载体,期望转化番茄开发新型宫颈癌治疗性疫苗-转基因植物口服疫苗.通过生物信息学分析HPV16 E6、E7基因,设计并合成密码子优化的靶基因E6-E7融合基因;并在目的基因的上游引入分子佐荆LTB基因,与Kozak序列等表达元件相偶联,以提高目的基因在植物表达系统的表达水平、增强其诱导黏膜免疫的免疫原性.目前已构建pX6-LTB-E7和pX6-LTB-E7-E6两个番茄转化双元载体.采用番茄Marker-Free系统转化和表达HPV16 E6、E7目的基因可以在转化后代中剔除标记基因,从而消除由标记基因可能引起的转基因植物口服疫苗的安全性问题,为HPV转基因植物口服疫苗应用奠定基础.     

Apoptosis induced by an antagonist peptide against HPV16 E7 in vitro and in vivo via restoration of p53

Human papilloma virus type 16 (HPV16) E7 is a viral oncoprotein that is believed to play a major role in cervical neoplasia. A novel antagonist peptide against HPV16 E7 was previously selected by phage display screening and the selected peptide was found to have anti-tumor efficacy against HPV16-positive cervical carcinoma through induction of cell cycle arrest. In the current study, to further elucidate the mechanisms of the antagonist peptide, the effects of the peptide on apoptosis are investigated by RT-PCR, Western blotting, MTT assay, TUNEL staining, Annexin V apoptosis assay, flow cytometry, and animal experiments. The antagonist peptide showed obvious anti-tumor efficacy through apoptosis induction, both in HPV16-positive cervical cancer cell lines and tumor xenografts. Our results also revealed that the peptide induced accumulation of cellular p53 and p21, and led to HPV16 E7 protein degradation. In the case of mRNA levels, it resulted in unaltered p53 and HPV16 E7 expression, but increased expression of p21. In contrast, the induction of apoptosis and p53 reactivation effects by the selected peptide were abolished after E7 knocked down with siRNA. These results demonstrate that the selected peptide can induce E7 degradation and lead to marked apoptosis in HPV16-related cancer cells by activating cellular p53 and its target genes, such as p21. Furthermore, the evident therapeutic efficacy obtained from the subcutaneous tumor model experiments in nude mice suggests a therapeutic potential for HPV16-related cancers of the selected peptide. Therefore, this specific peptide may be used to create specific biotherapies for the treatment of HPV 16-positive cervical cancers.     

Vesicular stomatitis virus-based therapeutic vaccination targeted to the E1, E2, E6, and E7 proteins of cottontail rabbit papillomavirus

Persistent human papillomavirus (HPV)-associated benign and malignant lesions are a major cause of morbidity and mortality worldwide. Vaccination against HPV early proteins could provide an effective means of treating individuals with established infections. Recombinant vesicular stomatitis virus (VSV) vectors have been used previously to elicit strong humoral and cellular immune responses and develop prophylactic vaccines. We have shown that VSV vectors also can be used to elicit therapeutic immunity in the cottontail rabbit papillomavirus (CRPV)-rabbit model of high-risk HPV infection. In the present study, three new VSV vectors expressing the CRPV E1, E2, or E7 protein were produced and compared to the previously generated VSV-E6 vector for therapeutic efficacy. To determine whether vaccine efficacy could be augmented by simultaneous vaccination against two CRPV proteins, the four vaccines were delivered individually and in all possible pairings to rabbits 1 week after CRPV infection. Control rabbits received the recombinant wild-type VSV vector or medium only. Cumulative papilloma volumes were computed for analysis of the data. The analyses showed that VSV-based vaccination against the E1, E2, E6, or E7 protein significantly reduced papilloma volumes relative to those of the controls. Furthermore, VSV-based CRPV vaccination cured all of the papillomas in 5 of 30 rabbits. Of the individual vaccines, VSV-E7 was the most effective. The VSV-E7 vaccine alone was the most effective, as it reduced cumulative papilloma volumes by 96.9% overall, relative to those of the controls, and ultimately eliminated all of the disease in all of the vaccinees. Vaccine pairing was not, however, found to be beneficial, suggesting antigenic competition between the coexpressed CRPV proteins. These preclinical results, obtained in a physiologically relevant animal model of HPV infection, demonstrate that VSV vectors deserve serious consideration for further development as therapeutic antitumor vaccines.     

Ubiquitin-fused and/or multiple early genes from cottontail rabbit papillomavirus as DNA vaccines

Human papillomavirus (HPV) vaccines have the potential to prevent cervical cancer by preventing HPV infection or treating premalignant disease. We previously showed that DNA vaccination with the cottontail rabbit papillomavirus (CRPV) E6 gene induced partial protection against CRPV challenge and that the vaccine's effects were greatly enhanced by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, two additional strategies for augmenting the clinical efficacy of CRPV E6 vaccination were evaluated. The first was to fuse a ubiquitin monomer to the CRPV E6 protein to enhance antigen processing and presentation through the major histocompatibility complex class I pathway. Rabbits vaccinated with the wild-type E6 gene plus GM-CSF or with the ubiquitin-fused E6 gene formed significantly fewer papillomas than the controls. The papillomas also required a longer time to appear and grew more slowly. Finally, a significant proportion of the papillomas subsequently regressed. The ubiquitin-fused E6 vaccine was significantly more effective than the wild-type E6 vaccine plus GM-CSF priming. The second strategy was to vaccinate with multiple CRPV early genes to increase the breadth of the CRPV-specific response. DNA vaccines encoding the wild-type CRPV E1-E2, E6, or E7 protein were tested alone and in all possible combinations. All vaccines and combinations suppressed papilloma formation, slowed papilloma growth, and stimulated subsequent papilloma regression. Finally, the two strategies were merged and a combination DNA vaccine containing ubiquitin-fused versions of the CRPV E1, E2, and E7 genes was tested. This last vaccine prevented papilloma formation at all challenge sites in all rabbits, demonstrating complete protection.     

HPV‐16 targeted DNA vaccine expression: The role of purification

DNA vaccines have come to light in the last decades as an alternative method to prevent many infectious diseases, but they can also be used for the treatment of specific diseases, such as cervical cancer caused by Human Papillomavirus (HPV). This virus produces E6 and E7 oncoproteins, which alter the cell cycle regulation and can interfere with the DNA repairing system. These features can ultimately lead to the progression of cervical cancer, after cell infection by HPV. Thus, the development of a DNA vaccine targeting both proteins arises as an interesting option in the treatment of this pathology. Nonetheless, before evaluating its therapeutic potential, the purity levels of a biopharmaceutical must meet the regulatory agency specifications. Previously, our research group successfully purified the supercoiled isoform of the recombinant HPV‐16 E6/E7 DNA vaccine with virtual 100% purity by affinity chromatography. The present work was designed to evaluate the effect that pDNA sample purity levels may exert in the expression of a target protein. Thus, in vitro studies were performed to assess the vaccine ability to produce the target proteins and to compare the expression efficiency between the pDNA sample obtained by affinity chromatography, which only presents the sc isoform and fulfils the regulatory agency recommendations, and the same DNA vaccine retrieved by a commercial purification kit, which contains different pDNA isoforms. Our achievements suggest that the E6/E7 DNA vaccine purified by affinity chromatography promotes higher E6 and E7 mRNA and protein expression levels than the DNA vaccine purified with the commercial kit. Overall, these results underline the importance that a purification strategy may present in the therapeutic outcome of recombinant DNA vaccines, envisaging their further application as biopharmaceuticals. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:546–551, 2018     

Bicistronic DNA Vaccines Simultaneously Encoding HIV,HSV and HPV Antigens Promote CD8+ T Cell Responses and Protective Immunity

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.     

Antitumor effect of therapeutic HPV DNA vaccines with chitosan-based nanodelivery systems

Background

Cervical cancer is the second-most-common cause of malignancies in women worldwide, and the oncogenic activity of the human papilloma virus types (HPV) E7 protein has a crucial role in anogenital tumors. In this study, we have designed a therapeutic vaccine based on chitosan nanodelivery systems to deliver HPV-16 E7 DNA vaccine, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer. We have developed a Nano-chitosan (NCS) as a carrier system for intramuscular administration using a recombinant DNA vaccine expressing HPV-16 E7 (NCS-DNA E7 vaccine). NCS were characterized in vitro for their gene transfection ability.

Results

The transfection of CS-pEGFP NPs was efficient in CHO cells and the expression of green fluorescent proteins was well observed. In addition, NCS-DNA E7 vaccine induced the strongest E7-specific CD8+ T cell and interferon γ responses in C57BL/6 mice. Mice vaccinated with NCS-DNA E7 vaccine were able to generate potent protective and therapeutic antitumor effects against challenge with E7-expressing tumor cell line, TC-1.

Conclusions

The strong therapeutic effect induced by the Chitosan-based nanodelivery suggest that nanoparticles may be an efficient carrier to improve the immunogenicity of DNA vaccination upon intramuscular administration and the platform could be further exploited as a potential cancer vaccine candidate in humans.     

表达CRT/E7融合蛋白的人乳头瘤病毒DNA疫苗对肿瘤生长和血管生成抑制作用的研究

研究HPV6b E7/CRT DNA疫苗免疫保护,清除已有感染和相关肿瘤细胞及其血管生成抑制作用,分析CRT抗血管生成的功能片段,为筛选高效的HPV疫苗提供实验依据。用重组质粒pcDNA3.1( )-GFP-CRT120/HPV6bE7、pcDNA3.1( )-GFP-HPV6bE7、pcDNA3.1( )-GFP-CRT120、pcDNA3.1( )-GFP-CRT180/HPV6bE7、pcDNA3.1( )-GFP-CRT180通过肌内注射途径免疫C57BL/6小鼠。Matrigel法进行抗血管活性检测;B16/HPV6bE7细胞接种于C57BL/6雌性小鼠建立荷瘤模型,观察各组DNA疫苗对HPV6bE7基因的荷瘤组织的出瘤时间和肿瘤大小的影响。结果显示:重组DNA疫苗pcDNA3.1-CRT180/HPV6bE7和pcDNA3.1-CRT180在动物体内能对bFGF诱导的新生血管的生成有明显的抑制作用;CRT180/HPV6bE7和CRT180能显著抑制荷瘤的大小且CRT180/HPV6bE7免疫组较其他组能明显延缓荷瘤的形成时间、生长速率以及肿瘤重量。CRT180/HPV6bE7免疫组较其他组能诱导更强的血管抑制作用和部分抑制肿瘤生长,推测抑制血管的功能片段存在于CRT 120~180 aa片段上。     

Transforming activity of a novel mutant of HPV16 E6E7 fusion gene

An optimized recombinant HPV16 E6E7 fusion gene(HPV16 ofE6E7)was constructed according to codon usage for mammalian cell expression,and a mutant of HPV16 ofE6E7 fusion gene(HPV16 omfE6E7)was generated by site-directed mutagenesis at L57G,C113R for the E6 protein and C24G,E26G for the E7 protein for HPV16 ofE6E7 [patent pending(CN 101100672)].The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice,but also maintained very good stability and antigenicity.These results suggests that the HPV16 omfE6E7 gene should undergo further study for application as a safe antigen-specific therapeutic vaccine for HPV16-associated tumors.     

RNA interference against HPV16 E7 oncogene leads to viral E6 and E7 suppression in cervical cancer cells and apoptosis via upregulation of Rb and p53

The simultaneous expression of human papillomavirus type 16 (HPV16) E6 and E7 oncogenes is pivotal for malignant transformation and maintenance of malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether HPV16 E6 and E7 could be both silenced to obtain most efficient antitumor activity by using RNA interference (RNAi) technology. Herein, we designed a small interfering RNA (siRNA) targeting HPV16-E7 region to degrade either E6, or truncated E6 (E6*) and E7 mRNAs and to simultaneously knockdown both E6 and E7 expression. Firstly, the sequence targeting HPV16-E7 region was inserted into the shRNA packing vector pSIREN-DNR, yielding pSIREN-16E7 to stably express corresponding shRNA. HPV16-transformed SiHa and CaSki cells were used as a model system; RT-PCR, Western Blotting, MTT assay, TUNEL staining, Annexin V apoptosis assay and flow cytometry were applied to examine the effects of pSIREN-16E7. Our results indicated that HPV16-E7 specific shRNA (16E7-shRNA) induced selective degradation of E6 and E7 mRNAs and proteins. E6 silencing induced accumulation of cellular p53 and p21. In contrast, E7 silencing induced hypophosphorylation of retinoblastoma (Rb) protein. The loss of E6 and E7 reduced cell growth and ultimately resulted in massive apoptotic cell death selectively in HPV-positive cancer cells, compared with the HPV-negative ones. We demonstrated that 16E7-shRNA can induce simultaneous E6 and E7 suppression and lead to striking apoptosis in HPV16-related cancer cells by activating cellular p53, p21 and Rb. Therefore, RNAi using E7 shRNA may have the gene-specific therapy potential for HPV16-related cancers.     

Enhanced anti-tumor effects of HPV16E749–57-based vaccine by combined immunization with poly(I:C) and oxygen-regulated protein 150

Background: It is well known that both heat shock protein (HSP) and Toll-like receptor (TLR)3 agonist polyinosinic:polycytidylic acid (poly(I:C)) are capable of promoting the antigen-specific immune responses. In the current study, we assessed whether the anti-tumor effects of the HPV16E7_49–57-based vaccine can be elevated by combined applications of poly(I:C) and oxygen-regulated protein 150 (ORP150) in a mouse cervical cancer model. Methods: Recombinant mouse ORP150 and HPV E7_49–57 peptide were combined to passively form the ORP150–E7_49–57 complex under heat shock conditions. The effects of ORP150–E7_49–57 complex plus poly(I:C) adjuvant on lymphocyte proliferation and functional cytotoxic T cells were investigated by methyl thiazolyl tetrazolium (MTT), ELISPOT, and non-radioactive cytotoxicity assays. Finally, the complex's therapeutic anti-tumor effects with and without adjuvant therapy were observed in a tumor challenge experiment. Results: This combination vaccine approach significantly enhanced the proliferation of splenocytes and induced strong E7_49–57-speci?c CTL responses. More importantly, the ORP150–E7_49–57 complex plus poly(I:C) vaccine format demonstrated more potent anti-tumor effects than ORP150–E7_49–57 complex alone or E7_49–57 plus poly(I:C) in TC-1 tumor-bearing mice. Conclusion: Both poly(I:C) and ORP150 chaperone can synergistically enhance the anti-tumor effects of the HPV16E7_49–57-based vaccine in vitro and in vivo. This strategy provides a platform for the design of a tumor therapeutic vaccine capable of inducing an effective anti-tumor immune response.     

HPV16+治疗性无佐剂蛋白疫苗—HPV16Z- Hsp65-E6/E7的构建、表达及纯化工艺研究

目的：研制针对我国宫颈癌高危相关的HPV16型的治疗性无佐剂蛋白疫苗。方法：应用PCR技术自我国山西宫颈癌高发现场分离到的毒株-HPV16z中获得E6/E7转化基因片段,自卡介苗菌株中克隆获得Hsp65基因片段,对E6/E7基因片段定点突变修饰,构建pET28a-Hsp65-E6/E7表达载体,在大肠杆菌BL21(DE3)中表达融合蛋白,并研究重组蛋白的纯化方案和工艺。结果：成功构建pET28a-Hsp65-E6/E7重组表达载体,E6/E7突变位点正确,融合蛋白在亲和层析柱上正确复性和初步纯化,经阴离子交换色谱纯化后蛋白纯度达到95%。结论：该研究为无佐剂治疗性重组蛋白疫苗Hsp65-E6/E7的进一步功能研究奠定了基础。