A novel therapeutic fusion protein vaccine by two different families of heat shock proteins linked with HPV16 E7 generates potent antitumor immunity and antiangiogenesis

Human papillomaviruses (HPV), particularly HPV16, is considered a necessary cause of cervical and oral cancer. Thus, the development of a therapeutic vaccine against HPV is important for the control of cervical cancer. However, therapeutic vaccination has been limited by inadequate antigen-specific immune responses. Heat shock proteins (HSP), including calreticulin (CRT), HSP70 and gp96, have been shown to act as potent immunoadjuvant to enhance antigen-specific tumor immunity. Previous studies have shown that N domain CRT (NCRT) or C-terminal half of HSP70 (hsp) linked with HPV16 E7 are capable of inducing potent antigen-specific CTL activity in experimental animal models. Here we developed a recombinant NCRT/E7/hsp fusion protein to investigate the synergistic effects of NCRT and hsp for enhancing the potency of HPV16 E7 therapeutic vaccine and evaluated the immune responses induced by this fusion protein. Our results demonstrated that NCRT and hsp synergistically exhibited significant increases in E7-specific CD8(+) T cell responses and impressive antitumor effects against E7-expressing tumors. Furthermore, the NCRT/E7/hsp fusion protein also generates potent antiangiogenic effects. These results indicate that NCRT/E7/hsp fusion protein is a promising therapeutic vaccine for treatment of cervical cancer through a combination of antigen-specific immunotherapy and antiangiogenesis, with possible therapeutic potential in clinical settings.     

Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX adjuvant in women with cervical intraepithelial neoplasia

PURPOSE: Persistent infection of cervical epithelium with "high risk" human papillomavirus (HPV) results in cervical intraepithelial neoplasia (CIN) from which squamous cancer of the cervix can arise. A study was undertaken to evaluate the safety and immunogenicity of an HPV16 immunotherapeutic consisting of a mixture of HPV16 E6E7 fusion protein and ISCOMATRIX adjuvant (HPV16 Immunotherapeutic) for patients with CIN. EXPERIMENTAL DESIGN: Patients with CIN (n = 31) were recruited to a randomised blinded placebo controlled dose ranging study of immunotherapy. RESULTS: Immunotherapy was well tolerated. Immunised subjects developed HPV16 E6E7 specific immunity. Antibody, delayed type hypersensitivity, in vitro cytokine release, and CD8 T cell responses to E6 and E7 proteins were each significantly greater in the immunised subjects than in placebo recipients. Loss of HPV16 DNA from the cervix was observed in some vaccine and placebo recipients. CONCLUSIONS: The HPV16 Immunotherapeutic comprising HPV16E6E7 fusion protein and ISCOMATRIX adjuvant is safe and induces vaccine antigen specific cell mediated immunity.     

Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX™ adjuvant in women with cervical intraepithelial neoplasia

Purpose: Persistent infection of cervical epithelium with “high risk” human papillomavirus (HPV) results in cervical intraepithelial neoplasia (CIN) from which squamous cancer of the cervix can arise. A study was undertaken to evaluate the safety and immunogenicity of an HPV16 immunotherapeutic consisting of a mixture of HPV16 E6E7 fusion protein and ISCOMATRIX™ adjuvant (HPV16 Immunotherapeutic) for patients with CIN.Experimental design: Patients with CIN (n = 31) were recruited to a randomised blinded placebo controlled dose ranging study of immunotherapy.Results: Immunotherapy was well tolerated. Immunised subjects developed HPV16 E6E7 specific immunity. Antibody, delayed type hypersensitivity, in vitro cytokine release, and CD8 T cell responses to E6 and E7 proteins were each significantly greater in the immunised subjects than in placebo recipients. Loss of HPV16 DNA from the cervix was observed in some vaccine and placebo recipients.Conclusions: The HPV16 Immunotherapeutic comprising HPV16E6E7 fusion protein and ISCOMATRIX™ adjuvant is safe and induces vaccine antigen specific cell mediated immunity.     

A novel multi-epitope vaccine of HPV16 E5E6E7 oncoprotein delivered by HBc VLPs induced efficient prophylactic and therapeutic antitumor immunity in tumor mice model

Human papilloma virus type 16 (HPV16) is the most prevalent etiologic agent associated with cervical cancer, and its early proteins E5, E6 and E7 play important roles in cervical epithelium transformation to cervical intraepithelial neoplasia and even cervical cancer. Hence, these oncoproteins are ideal target antigens for developing immunotherapeutic vaccines against HPV-associated infection and cervical cancer. Currently, multi-epitope vaccines have been a promising strategy for immunotherapy for viral infection or cancers. In this study, the E5aa28-46, E6aa37-57 and E7aa26-57 peptides were selected and linked to form a novel multi-epitopes vaccine (E765m), which was inserted into the major immune dominant region (MIR) of hepatitis B virus core antigen (HBc) to construct a HBc-E765m chimeric virus-like particles (cVLPs). The immunogenicity and immunotherapeutic effect of the cVLPs vaccine was evaluated in immunized mice and a tumor-bearing mouse model. The results showed that HBc-E765m cVLPs elicited high E5-, E6- and E7- specific CTL and serum IgG antibody responses, and also relatively high levels of the cytokines IFN-γ, IL-4 and IL-5. More importantly, the cVLPs vaccine significant suppressed tumor growth in mice bearing E5-TC-1 tumors. Our findings provide strong evidence that this novel HBc-E765m cVLPs vaccine could be a candidate vaccine for specific immunotherapy in HPV16-associated cervical intraepithelial neoplasia or cervical cancer.     

Optimal induction of HPV DNA vaccine-induced CD8 T cell responses and therapeutic antitumor effect by antigen engineering and electroporation

Since human papillomavirus (HPV) E6 and E7 are promising tumor antigens, we engineered E6 and E7 antigens to generate an optimal HPV DNA vaccine by codon optimization (Co), fusion of E6 and E7, addition of a tissue plasminogen activator (tpa) signal sequence, addition of CD40 ligand (CD40L) or Fms-like tyrosine kinase-3 ligand (Flt3L). The resulting constructs were investigated in terms of their antitumor activity as well as induction of HPV-specific CD8⁺ T cell responses. When E6^Co and E7^Co were fused (E67^Co), CD8⁺ T cell responses specific for E6 or E7 antigen decreased, but the preventive antitumor effect rather improved, demonstrating the importance of broad immunity. Interestingly, Flt3L-fused HPV DNA vaccine exhibited stronger E6- and E7-specific CD8⁺ T cell responses as well as therapeutic antitumor effect than that of CD40L linked HPV DNA vaccine. Finally, the optimal construct, tFE67^Co, was generated by including tpa signal sequence, Flt3L, fusion of E6 and E7, and codon optimization, which induces 23 and 25 times stronger E6- and E7-specific CD8⁺ T cell responses than those of initial E67 fusion construct. In particular, inclusion of electroporation in intramuscular immunization of tFE67^Co further enhances HPV-specific CD8⁺ T cell responses, leading to complete tumor regression in a therapeutic setting. Thus, our results provide valuable insight on effective HPV DNA vaccine design and suggest that tFE67^Co delivered with electroporation may be a promising therapeutic HPV DNA vaccine against cervical cancer.     

Pre-clinical safety and efficacy of TA-CIN, a recombinant HPV16 L2E6E7 fusion protein vaccine, in homologous and heterologous prime-boost regimens

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical intraepithelial neoplasia (CIN) and cancer. A newly designed vaccine, comprising the HPV16 L2, E6 and E7 as a single fusion protein (TA-CIN), was shown to elicit HPV16-specific CTL, T-helper cells and antibodies in a pre-clinical mouse model. These immune responses effectively prevented outgrowth of HPV16-positive tumour cells in a prophylactic setting as well as in a minimal residual disease setting. CTL immunity was optimally induced when TA-CIN was employed in heterologous prime-boost regimens in combination with TA-HPV, a clinical grade vaccinia-based vaccine. These data provide a scientific basis for the use of TA-CIN, alone or in combination with TA-HPV in future human trials.     

HPV E6/E7研究现状及临床展望

人乳头状瘤病毒是宫颈癌最重要的致病因素,其早期蛋白E6/E7的持续表达可致细胞p53、pRb蛋白缺失或失活,并通过多种机制与其他细胞因子相互作用,促使细胞发生癌变。其mRNA表达水平提示病毒转录活性,可能具有诊断和预测宫颈病变的价值,因此有研究应用HPV E6/E7 mRNA筛查不同人群宫颈病变并随访、预测病变发生风险,但各研究结果不一,不同mRNA检测方法也是影响研究结果的因素之一。HPV E6/E7蛋白检测在临床应用较少,而以HPV E6/E7为靶点的宫颈癌免疫治疗已成为研究的热点,在动物实验及临床前研究中取得了一定进展。     

Antitumor efficacy of Venezuelan equine encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes

An effective vaccine for treating human papillomavirus (HPV)-associated malignancies such as cervical cancer should elicit strong T cell-mediated immunity (CMI) against the E6 and/or E7 proteins necessary for the malignant state. We have developed Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) vaccines encoding the HPV16 E6 and E7 genes and tested their immunogenicity and antitumor efficacy. The E6 and E7 genes were fused to create one open reading frame and mutated at four or at five amino acid positions to inactivate their oncogenic potential. VRP encoding mutant or wild type E6 and E7 proteins elicited comparable cytotoxic T lymphocyte (CTL) responses to an immunodominant E7(49-57) epitope and generated comparable antitumor responses in several HPV16 E6(+)E7(+) tumor challenge models: protection from either C3 or TC-1 tumor challenge was observed in 100% of VRP-vaccinated mice. Eradication of C3 tumors was observed in approximately 90% of mice following therapeutic VRP vaccination. Eradication of HLF16 tumors lacking the E7(49-57) epitope was observed in 90% of human leukocyte antigen (HLA)-A(*)0201 transgenic mice following therapeutic VRP vaccination. Finally, the predicted inactivation of E6 and E7 oncogenic potential was confirmed by demonstrating normal levels of both p53 and retinoblastoma proteins in human mammary epithelial cells (MEC) infected with VRP expressing mutant E6 and E7 genes. These promising results support the continued development of mutant E6 and E7 VRP as safe and effective candidates for clinical evaluation against HPV-associated disease.     

Immunization of protein HPV16 E7 in fusion with mouse HSP70 inhibits the growth of TC-1 cells in tumor bearing mice

Human papillomavirus (HPV) 16 is the primary etiologic agent of cervical cancer. Most HPV16 therapeutic vaccines target E7 protein which is consistently expressed in tumor cells. In this study, we cloned mouse autologous heat shock protein 70 (mHSP70) gene from mouse liver cells and then expressed mHSP70 and fused HPV16 E7-mHSP70 (E7 at the N-terminus and mHSP70 at the C-terminus) proteins in E. coli. Then we investigated the inhibition of TC-1 cell growth by using the E7-expressing murine tumor cell line, TC-1, as a model of cervical cancer. In this model, mice were immunized with the fusion protein of E7-mHSP70 without any adjuvant. The results showed that prophylactic immunization of E7-mHSP70 protected mice against challenge with TC-1 cells. In addition, therapeutic immunization with E7-mHSP70 could inhibit TC-1 tumor growth on lungs. Our study demonstrated that immunization with E7-mHSP70 protein without any adjuvant could generate anti-tumor effect in mice challenged with TC-1 cells.     

Generation and characterization of a preventive and therapeutic HPV DNA vaccine

Cervical cancer is one of the most common cancers in women worldwide. Persistent infection with human papillomavirus (HPV) is considered to be the etiological factor for cervical cancer. Therefore, an effective vaccine against HPV infections may lead to the control of cervical cancer. An ideal HPV vaccine should aim to generate both humoral immune response to prevent new infections as well as cell-mediated immunity to eliminate established infection or HPV-related disease. In the current study, we have generated a potential preventive and therapeutic HPV DNA vaccine using human calreticulin (CRT) linked to HPV16 early proteins, E6 and E7 and the late protein L2 (hCRTE6E7L2). We found that vaccination with hCRTE6E7L2 DNA vaccine induced a potent E6/E7-specific CD8+ T cell immune response, resulting in a significant therapeutic effect against E6/E7 expressing tumor cells. In addition, vaccination with hCRTE6E7L2 DNA generated significant L2-specific neutralizing antibody responses, protecting against pseudovirion infection. Thus, the hCRTE6E7L2 DNA vaccines are capable of generating potent preventive and therapeutic effects in vaccinated mice. Our data has significant clinical implications.     

Clinical studies of human papilloma vaccines in pre-invasive and invasive cancer

Cervical cancer is the second most common cause of cancer death in women worldwide. It is almost invariably associated with infection with human papilloma virus (HPV) particularly types 16 and 18. The ubiquitous expression of E6 and E7 oncogene products has been recognised as an attractive target for CTL-mediated immunotherapy. In-vivo expansion of an HPV oncogene product specific MHC class 1 restricted response has been demonstrated using intradermally administered live vaccinia virus HPV 16 and 18 E6/E7 gene construct (TA-HPV, Cantab Pharmaceuticals). Responses have been seen in 1/3 evaluable patients with advanced cervical cancer, and 3/12 CIN3 volunteers, and in 4/29 patients with early invasive cervical cancer (Rankin et al. Proceedings of 91st AACR Meeting, San Francisco, April 2000). In addition, the adoptive transfer of ex vivo HPV 16 or 18 positive autologous tumour lysate pulsed dendritic cells is currently being tested as an alternative means of expanding HPV specific CTL in advanced cervical cancer patients. So far an HLA-A*O201 restricted CD8 T cell response has been recorded in the single HLA-A*O201 patient whose tumour was shown to be HPV16 positive. It appears therefore feasible to induce HPV specific CTL responses in patients with cervical cancer using several vaccine strategies. However, further clinical trials are needed to determine the full anti-tumour potential of this vaccine based immunotherapy.     

ISCOMATRIX adjuvant: an adjuvant suitable for use in anticancer vaccines

Human Papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are associated with cervical cancer development and progression and can therefore be used as target antigens for cancer immunotherapy. In this study we evaluated the immunogenicity in mice, of different vaccine formulations using recombinant HPV16 derived E6E7 or E7GST fusion proteins. When co-administered with ISCOMATRIX adjuvant, these E6E7 proteins consistently induced E7 specific CTL, in vivo tumor protection, antibody and DTH responses. ISCOMATRIX adjuvant has been developed for use in the formulation of novel human vaccines and has been evaluated for safety and toxicity in human trials. A formulation containing aluminum hydroxide (Al(OH)3) gave a lesser degree of E7 specific antibody, and no local E7 specific CTL response but similar DTH and tumor protection. These findings demonstrate the potential of ISCOMATRIX adjuvant to stimulate both cellular and humoral immune responses to endogenously processed target antigens, and hence is the preferred adjuvant when CTL responses are desirable.     

Effect of TA-CIN (HPV 16 L2E6E7) booster immunisation in vulval intraepithelial neoplasia patients previously vaccinated with TA-HPV (vaccinia virus encoding HPV 16/18 E6E7)

Heterologous prime-boost vaccination schedules employing TA-HPV, a vaccinia virus encoding HPV 16/18 E6 and E7, in combination with TA-CIN, an HPV 16 L2E6E7 fusion protein, may offer advantages over the use of either agent alone for the immunotherapy of human papillomavirus (HPV) type 16-associated vulval intraepithelial neoplasia (VIN). In the present study, 10 women with HPV 16-positive high grade VIN, previously primed with TA-HPV, received three booster immunisations with TA-CIN. All but one demonstrated HPV 16-specific proliferative T-cell and/or serological responses following vaccination. Three patients additionally showed lesion shrinkage or symptom relief, but no direct correlation between clinical and immunological responses was seen.     

人乳头瘤病毒16 E2、E6癌基因在子宫颈癌中的表达及E2缺失分析

目的探讨人乳头瘤病毒（HPV）16型E2、E6癌基因的表达及含量变化在子宫颈癌发生中的作用，以及E2基因的缺失与子宫颈病变的关系。方法收集山西省肿瘤医院经病理学确诊的141例子宫颈癌新发病例作为病例组，同时选择在同一医院住院、经组织病理学确诊的137例子宫肌瘤患者为良性肿瘤对照组，从社区妇科体检的1582名妇女中随机抽取129名妇女为健康对照组。利用多重PCR对从子宫颈组织和脱落细胞中提取的DNA进行HPVl6 E2、E6基因扩增，在VILBER凝胶成像仪上利用Bio-ID＋＋软件分析E2、E6基因的相对含量。结果HPVl6E6阳性率在子宫颈癌组、子宫肌瘤组和健康对照组分别为46．8％、24．1％和2．3％，HPVl6E2阳性率分别为36．2％、19．0％和0．0％，差异均有统计学意义。子宫颈癌组和子宫肌瘤组中E2缺失率分别为22．73％和21．21％，健康对照组未检出E2阳性者。HPVl6E6和E2含量均为病例组高于对照组，在子宫颈癌组内E6含量明显高于E2（t=2．70，P=0．009），两者的比值为1．24。结论子宫颈癌患者HPVl6E2、E6癌基因的阳性表达率和表达量均高于子宫肌瘤患者和健康妇女，E6癌基因的高表达在子宫颈癌发生中起重要作用，而E2基因的缺失在子宫颈癌变过程中可能发挥着不可忽视的作用。     

HPV 18型E6蛋白抗原表位预测与分析

目的 用生物信息学方法分析预测HPV 18型E6蛋白的B细胞和T细胞表位,为宫颈癌免疫治疗的疫苗及药物开发提供参考靶点。方法 基于NCBI中HPV18型E6蛋白的氨基酸序列,利用DNA star软件预测其二级结构、亲水性、表面可及性及柔韧性;利用ABCpred软件预测出其B细胞表位;综合使用CTLPred和NetMHC 4.0软件分析其CTL表位;ProPred和NetMHCIIpan 3.1软件预测其Th表位。结果 HPV 18型E6蛋白二级结构中α螺旋和β转角较多且分布较为均匀;预测出了4个B细胞表位,它们大多与亲水性强、表面可及性好、柔韧性好的蛋白区域重叠,易于结合抗体;E6蛋白具有丰富的细胞毒性和辅助性T细胞表位。结论 HPV 18型E6蛋白作为宫颈癌免疫治疗的理想靶点具有同时诱导特异性体液免疫和细胞免疫的潜能,通过对其B/T细胞抗原表位的预测为免疫治疗相关疫苗及药物抗原肽的选择提供了理论依据。     

Serum One-Carbon Metabolites and Risk of Cervical Cancer

Most cases of cervical cancer are associated with human papilloma virus (HPV) infection of high risk types. In folate deficiency, heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) interferes with HPV16 viral capsid protein synthesis. We aimed to study the importance of 1-carbon metabolism in cervical carcinogenesis by examining serum vitamin B₁₂ (cobalamin), homocysteine, folate levels, and the RNA and protein expression of HPV16 L1, L2, E6, E7, and to correlate them with hnRNP-E1 expression and HPV infection in normals, squamous intraepithelial lesions (SILs), and cervical cancer subjects. Serum cobalamin, folate, and homocysteine were estimated using kits, RNA by real time PCR and proteins by Western blotting. We observed that lower folate and vitamin B₁₂ levels were associated with HPV infection. hnRNP-E1 progressively decreased from normals (100%) to SILs (75%) to cervical cancer (52.6%). The findings show that HPV16 E6 and E7 are overexpresed whereas HPV16 L1 and L2 are downregulated at mRNA and protein levels in cervical cancer as compared to normals and SILs. The results indicate that perhaps the reduced expression of hnRNP-E1 might be involved with the cervical cancer pathogenesis, with folate playing a role in the natural history of HPV infection.     

Characterization of HPV18 E6-specific T cell responses and establishment of HPV18 E6-expressing tumor model

Human papillomavirus (HPV) has been identified as the primary etiologic factor of cervical cancer, and subsets of anogenital and oropharyngeal cancers. HPV18 is the second most prevalent high-risk HPV type after HPV16. Furthermore, HPV18 is responsible for approximately 12% of cervical squamous cell carcinoma and 37% of cervical adenocarcinoma cases worldwide. In this study, we aimed to characterize the HPV18-E6-specific epitope and establish an HPV18 animal tumor model to evaluate the E6-specific immune response induced by our DNA vaccine. We vaccinated naïve C57BL/6 mice with a prototype DNA vaccine, pcDNA3-HPV18-E6, via intramuscular injection followed by electroporation, and analyzed the E6-specific CD8+ T cell responses by flow cytometry using a reported T cell epitope. We then characterized the MHC restriction element for the characterized HPV18-E6 epitope. Additionally, we generated an HPV18-E6-expressing tumor cell line to study the antitumor effect mediated by E6-specific immunity. We observed a robust HPV18-E6aa67-75 peptide-specific CD8+ T cell response after vaccination with pcDNA3-HPV18-E6. Further characterization demonstrated that this epitope was mainly restricted by H-2K^b, but was also weakly presented by HLA-A¹0201, as previously reported. We observed that vaccination with pcDNA3-HPV18-E6 significantly inhibited the growth of HPV18-E6-expressing tumor cells, TC-1/HPV18-E6, in mice. An antibody depletion study demonstrated that both CD4+ and CD8+ T cells are necessary for the observed antitumor immunity. The characterization of HPV18-E6-specific T cell responses and the establishment of HPV18-E6-expressing tumor cell line provide infrastructures for further development of HPV18-E6 targeted immunotherapy.     

Intranasal immunization with synthetic peptides corresponding to the E6 and E7 oncoproteins of human papillomavirus type 16 induces systemic and mucosal cellular immune responses and tumor protection

The E6 and E7 oncoproteins of the high-risk HPV type16 represent ideal targets for HPV vaccine development, they being consistently expressed in cervical cancer lesions. Since HPV-16 is primarily transmitted through genital mucosal route, mucosal immune responses constitute an essential feature for vaccination strategies against HPV-associated lesions. We present here evidence showing that mucosal immunization of mice by the intranasal route with a mixture of peptides E7(44-62) and E6(43-57) from the E7 and E6 oncoproteins of HPV-16, respectively, using a mutant cholera toxin adjuvant (CT-2*), primed strong antigen-specific cellular immune responses in systemic and mucosal tissues. Significant levels of IFN-gamma production by both CD4 and CD8 cells were observed along with CTL responses that were effective against both peptide-pulsed targets as well as syngeneic tumor cells (TC-1) expressing the cognate E6 and E7 proteins. Furthermore, mice immunized with the peptide mixture and CT-2* effectively resisted TC-1 tumor challenge. These results together with our earlier observations that T cell responses to these peptides correlate with recurrence-free survival in women after ablative treatment for HPV-associated cervical intraepithelial neoplasia, support the potential of these E6 and E7 peptides for inclusion in vaccine formulations.