Comparison of human papillomavirus type 16 L1 chimeric virus-like particles versus L1/L2 chimeric virus-like particles in tumor prevention

Chimeric human papillomavirus (HPV) virus-like particles (cVLPs) with the HPV16 E7 antigen fused to either the major capsid protein, L1, or the minor capsid protein, L2, have been used independently to protect against the formation of HPV-induced tumors in animal models. However, the advantages and disadvantages of both types of particles with respect to production and vaccine efficacy have never been analyzed. Therefore, in this study, we compared cVLPs with the HPV16 E7 antigen fused to L1 versus cVLPs with E7 fused to L2 with respect to their ability to protect mice from tumor challenge. The first 57 amino acids of E7 were used to overcome the size limitation and limited VLP production imposed by inserting polypeptides into L1 cVLPs. C57BL/6 mice were immunized with the above cVLPs at various doses. Tumor challenge was then performed with HPV16 E7-positive TC-1 cells. HPV16 L1-E7((1-57)) was superior to HPV16 L1/L2-E7((1-57)) in eliciting tumor protection at equivalent doses, although both types of particles were able to protect mice. Both cVLPs induced a specific cytotoxic T lymphocyte (CTL) response to the H2-D(b)-restricted E7 peptide (E7(49-57)) as determined by an ELISPOT assay and tetramer staining; however, immunization with the L1-E7((1-57)) cVLPs resulted in twofold higher CTL precursor frequencies. Our results demonstrate that cVLPs with the antigen fused to L1 are a more efficient vaccine with respect to tumor prevention than cVLPs with the antigen fused to L2. At the same time, however, L1 cVLPs are limited by the size of the antigen that can be incorporated and in the amount of cVLP that can be obtained from cultures when compared to L1/L2 cVLPs. This balances out their superior ability to induce protective immunity.     

Comparison of DNA- and mRNA-transfected mouse dendritic cells as potential vaccines against the human papillomavirus type 16 associated oncoprotein E7

BACKGROUND: Dendritic cells (DCs) mediate the generation of strong cytotoxic T-lymphocyte (CTL) responses by functioning in antigen presentation and exerting adjuvant properties. We compared several activation markers and parameters of biological activity of DNA- and mRNA-transfected DCs in vitro and in vivo. METHODS: CpG-matured, bone marrow derived C57BL/6 mouse DCs were electroporated either with enhanced green fluorescence protein (EGFP) or human papillomavirus type 16 (HPV16) E7 expression plasmids or in vitro transcribed mRNAs encoding for the codon-optimized E7 or a shuffled version thereof. Activation marker expression and antigen presentation was analysed by fluorescence-activated cell sorting. The migratory behaviour of transfected DCs were investigated by in vitro chemotaxis experiments and cytokine expression by ELISA. CTL-priming capacity of transfected DCs were determined by vaccination of mice. RESULTS: mRNA transfection produced a two- to fourfold increase of the activation markers CD40, CD80, CD86 and MHC I and MHC II molecules. Predominately antigen-expressing DCs migrated after mRNA transfection. Furthermore, mRNA-transfected DCs were capable of inducing a chemokine gradient. After maturation, electroporation and activation with soluble CD40 ligand and interferon-y, DCs displayed a T-helper cell type 2 cytokine expression pattern. Nevertheless, E7-transfected DCs were able to prime E7-specific CTL responses in vivo. The highest E7-specific CTL frequencies were found in mice immunized with mRNA-transfected DCs. The in vitro expanded CTLs exerted functional E7-specific cytotoxic activity. CONCLUSIONS: Genetically modified DCs are suitable vehicles for the induction of E7-specific CTL responses in mice and hence could help to eradicate HPV-associated lesions in humans.     

Targeting human papillomavirus type 16 E7 to the endosomal/lysosomal compartment enhances the antitumor immunity of DNA vaccines against murine human papillomavirus type 16 E7-expressing tumors

DNA vaccination is an attractive approach for tumor immunotherapy because of its stability and simplicity of delivery. Advances demonstrate that helper T cell responses play a critical role in initiating immune responses. The aim of the current study is to test whether targeting HPV-16 E7 to the endosomal/lysosomal compartment can enhance the potency of DNA vaccines. We linked the lysosome-associated membrane protein 1 (LAMP-1) to HPV-E7 to construct a chimeric DNA, Sig/E7/LAMP-1 DNA. For in vivo tumor prevention experiments, mice were vaccinated with E7 DNA or Sig/E7/LAMP-1 DNA via gene gun, followed by tumor challenge. For in vivo tumor regression experiments, mice were first challenged with tumor cells and then vaccinated with E7-DNA or Sig/E7/LAMP-1 DNA. Intracellular cytokine staining with flow cytometry analysis, cytotoxic T lymphocyte (CTL) assays, enzyme-linked immunoabsorbent assay (ELISA), and enzyme-linked immunospot (ELISPOT) assays were used for in vitro E7-specific immunological studies. In both tumor prevention and tumor regression assays, Sig/E7/LAMP-1 DNA generated greater antitumor immunity than did wild-type E7 DNA. In addition, mice vaccinated with Sig/E7/LAMP-1 DNA had greater numbers of E7-specific CD4+ helper T cells, higher E7-specific CTL activity, and greater numbers of CD8+ T cell precursors than did mice vaccinated with Sig/E7 or wild-type E7 DNA. Sig/E7 generated a stronger E7-specific antibody response than did Sig/E7/LAMP-1 or wild-type E7 DNA. Our results indicate that linkage of the antigen gene to an endosomal/lysosomal targeting signal may greatly enhance the potency of DNA vaccines.     

Selective expression of immunogenic,virus-like particle-derived antibody-binding epitopes

The incorporation of linear and conformational antibody-binding epitopes into polyepitope, chimeric antigens with satisfactory immunogenicity is a challenge. We selectively expressed antigen fragments encoding the linear e2 epitope (C(79-149)) of hepatitis B virus (pre)core antigen (HBc/eAg) and the conformational 'a' epitope (S(80-180)) of hepatitis B surface antigen (HBsAg) in a novel system. The domains were expressed as chimeric antigens containing either heat shock protein (hsp)73-binding simian virus 40 large tumor antigen (e.g. T(77)) or non-hsp-binding (e.g. T(60)) sequences at their N-termini. We compared their type of expression with their immunogenicity for B cells (when delivered as a DNA vaccine). The type of expression investigated included their level of expression, the secretion or intracellular expression of the antigen and the stress protein (hsp)-associated versus nonassociated expression. The linear e2 epitope of HBc/eAg was efficiently expressed as an intracellular, hsp73-binding fusion protein, and efficiently primed an HBc/eAg-specific antibody response when delivered in this form. The conformational 'a' epitope of HBsAg most efficiently stimulated B cells as a secreted, non-hsp-associated fusion protein. These data demonstrate that different B cell-stimulating epitopes of vaccine-relevant viral antigens can be selectively isolated and expressed in suitable expression systems, but that the requirements that have to be fulfilled to obtain optimal immunogenicity differ strikingly between individual epitopes.     

Induction of cellular immunosuppression by the human papillomavirus type 16 E7 oncogenic protein.

The human papillomavirus type 16 (HPV-16) E7 oncogenic protein is found in the culture supernatant of SiHa cells, a cervical carcinoma cell line. Extracellular E7 protein, acting as a viral toxin in human immune cells, induces the overproduction of the immune suppressive IFN alpha cytokine by APCs, and inhibits the T-cell response to recall and allogenic antigens. These effects should be taken into account for the design of anti-human cervical carcinoma vaccines.     

A combination of DNA vaccines targeting human papillomavirus type 16 E6 and E7 generates potent antitumor effects

Human papillomavirus (HPV) infects large numbers of women worldwide and is present in more than 99% of all cervical cancers. HPV E6 and E7 are two viral oncoproteins that are consistently expressed in HPV infections and HPV-associated malignancies. We have previously developed DNA vaccines encoding calreticulin (CRT) linked either to HPV type 16 (HPV-16) E6 or to HPV-16 E7, both of which generated significant antitumor effects against E6- and E7-expressing tumors. In this study, we demonstrate that simultaneous vaccination of C57BL/6 mice or HLA-A2 transgenic mice with both CRT/E6 and CRT/E7 DNA vaccines generates significant E6- and E7-specific T-cell immune responses in vaccinated mice. Furthermore, combined vaccination with both CRT/E6 and CRT/E7 DNA generates significantly better therapeutic antitumor effects against HPV E6- and E7-expressing tumors than vaccination with either CRT/E6 DNA or CRT/E7 DNA alone. Our data suggest that it may be desirable to combine DNA vaccines targeting E6 with DNA vaccines targeting E7 to develop effective immunotherapeutic strategies for control of HPV infection and HPV-associated lesions in a clinical setting.     

耐核糖核酸酶病毒样颗粒的构建和表达

目的：为耐核糖核酸酶（RNase)的RNA标准品和质控品的表达制备提供1个通用载体平台。方法：将MS2噬菌体基因组中成熟酶蛋白和包膜蛋白基因编码序列的1．7kb cDNA目的片段，用Hind Ⅲ和EcoR I酶切后，用于相同内切酶酶切的表达载体质粒PET28b中，并在T4 DNA连接酶的存在下连接，构建一新的表达载体pI NCCL,再转化BL21－DE3 E.Coli进行原核表达。结果：成功构建出新的表达载体pI NCCL,经原核表达为耐RNase的病毒样颗粒。结论：本研究得到的pI NCCL表达载体及原核表达系统，可作为1个耐RNase的RNA标准品与质控品的构建和制备表达通用载体平台，以促进有关标准品和质控品的研究。     

Cervarix--a bivalent L1 virus-like particle vaccine for prevention of human papillomavirus type 16- and 18-associated cervical cancer

Cervical cancer continues to have a devastating impact on women worldwide. An estimated 470,000 women are diagnosed with the disease every year and, of these, > 230,000 die from it. Recent decades have witnessed considerable advances in our understanding of cervical carcinogenesis, in particular the causal role that oncogenic human papillomavirus infection plays in its etiology. In countries that lack organized cervical screening programs, prophylactic vaccination against human papillomavirus may offer the most effective way of reducing mortality from the disease. Cervarix, a bivalent L1 virus-like particle vaccine targeted against human papillomavirus types 16 and 18, has been developed to address this possibility. The clinical experience of Cervarix and its potential role in reducing the global burden of cervical cancer is discussed.     

A therapy modality using recombinant IL-12 adenovirus plus E7 protein in a human papillomavirus 16 E6/E7-associated cervical cancer animal model

Interleukin (IL)-12 has been reported to induce cellular immune responses for protection against tumor formation. Here we investigate the utility of adenoviral delivery of IL-12 as an adjuvant for a human papillomavirus E7 subunit vaccine in a mouse tumor challenge model. Direct intratumoral injection of AdIL-12 resulted in a significant suppression of tumor growth compared to the control group. Injection of E7 protein into either a tumor site or the distance site along with AdIL-12 further enhanced antitumor effects significantly higher than either AdIL-12 or E7 injection alone. This combined injection resulted in complete regression of 9-mm-sized tumor in 40% of animals as well as lasting antitumor immunity against tumor recurrence. We also evaluated immune responses induced by these injections. AdIL-12 plus E7 enhanced E7-specific antibody responses significantly higher than AdIL-12 or E7 injection. In particular, the production level of interferon (IFN)-gamma from E7-specific CD4(+) T cells was similar between AdIL-12 group and AdIL-12 + E7 group. However, IFN-gamma production from E7-specific CD8(+) T cells was the most significant when injected with AdIL-12 + E7. This was consistent with intracellular IFN-gamma staining levels of CD8(+) T cells, suggesting that AdIL-12 + E7 injection enhances antitumor immunity in the human papillomavirus (HPV) 16 tumor model through increased expansion of the cytotoxic T-lymphocyte (CTL) subset. This enhanced protection appeared to be mediated by CD8(+) T cells, as determined by in vivo T-cell subset deletion. Thus, these studies demonstrate that E7 vaccines can induce CTL responses responsible for antitumor effects in the presence of IL-12 delivered via adenovirus vectors. This likely provides one additional approach for immune therapy against cervical cancers.     

Human papillomavirus type 16 L1E7 chimeric capsomeres have prophylactic and therapeutic efficacy against papillomavirus in mice

Genital human papillomavirus (HPV) infection is the primary cause of cervical cancer in women. Although the HPV recombinant L1 protein was recently licensed as an available vaccine, it has numerous shortcomings. New vaccination strategies should be considered. To enable the design of a prophylactic and therapeutic low-cost vaccine candidate, chimeric HPV16 L1DeltaC34E7N1-60 capsomeres were produced in Escherichia coli. The immune characteristics and potential prophylactic and therapeutic effects of these capsomeres were examined in C57BL/6 mice. Following protein purification and renaturation, the majority of the recombinant chimeric proteins (L1DeltaC34E7N1-60) assembled into capsomeres. These capsomeres were able to induce conformational and neutralizing antibodies against HPV virus-like particles and trigger cell-mediated specific immune responses against the L1 and E7 peptides. In vivo tumor challenge assays showed that mice immunized with the capsomeres were protected against a challenge with both C3 and TC-1 tumor cells. Furthermore, in vivo tumor rejection assays showed that capsomeres have therapeutic efficacy in mice following inoculation with C3 and TC-1 tumor cells. Chimeric capsomeres are capable of preventing and eliminating HPV16 infection. Therefore, our study has provided an economical vaccine candidate.     

高危型人乳头状瘤病毒E6/E7mRNA检测对宫颈病变的诊断研究

目的探讨高危型人乳头状瘤病毒E6/E7 mRNA检测对宫颈病变的诊断价值。方法选择我院2019年3月-11月接诊的疑似宫颈病变患者,筛选163例患者,采用电脑随机分组的方式,163例分为81例对照组以及82例研究组,对照组接受高危型人乳头瘤病毒(human papollomavirus,HPV)DNA检测,研究组患者均接受高危型人乳头状瘤病毒E6/E7 mRNA检测,对比两组患者的检测准确率。结果研究组慢性宫颈炎、宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)I型宫颈癌诊断准确率均高于对照组,有统计学意义(P<0.05);研究组与对照组在CIN Ⅱ型、CIN Ⅲ型诊断准确率上虽无统计学意义,但是研究组的准确率高于对照组。结论对于疑似宫颈病变的患者而言,接受高危型人乳头状瘤病毒E6/E7 mRNA检测,不仅能够有效的判断疾病的类型,且诊断准确率较高,值得推广。     

Characterization of in vivo expression of the human papillomavirus type 16 E4 protein in cervical biopsy tissues.

The role of human papillomavirus (HPV) proteins in the pathogenesis of cervical intra-epithelial neoplasia (CIN) and invasive cervical cancer is poorly understood. To characterize E4 protein expression in 49 paraffin-embedded cervical biopsies representing different histopathologic grades of disease, antibodies were elicited to a synthetic peptide corresponding to amino acids 20-34 of a protein predicted to be encoded by the HPV 16 E4 open reading frame. The E4 protein was detected throughout the spectrum of CIN, from CIN1 to CIN3. Expression was localized to the cell nucleus, primarily in the superficial layers of the squamous cervical epithelium. Ultrastructural studies showed that the E4 protein was organized into compact, intranuclear arrays 25-35 nm in diameter. E4 protein expression was also demonstrated in some histologically normal tissues containing HPV 16 DNA, but not in any of five cervical cancers containing HPV 16 DNA. These results suggest that E4 protein expression may precede development of light microscopic tissue abnormalities, that it may continue through the spectrum of CIN, and that expression of this protein may be reduced or terminated in invasive cancer. The function of this protein remains unknown, but its nuclear localization may be consistent with a role in viral maturation.     

T cell response to human papillomavirus 16 E7 in mice: comparison of Cr release assay,intracellular IFN-gamma production,ELISPOT and tetramer staining

Successful vaccination against infections by high-risk papillomaviruses aiming at the prevention of cervical cancer most likely requires the induction of neutralizing antibodies and human papillomavirus (HPV)-specific T cells directed against early viral proteins such as E7. Whereas the technology for detection of antibodies is well established, measurement of T cells is more cumbersome and standardization of assays is difficult. By using chromium release assay, ELISPOT, tetramer staining and intracellular IFN-gamma assay, we compared the levels of HPV 16 E7-specific T cells obtained after immunization of C57BL/6 mice with different DNA expression vectors. We found that all four assays gave highly comparable results. ELISPOT can be recommended for future studies as it indicates the presence of activated (i.e. IFN-gamma-secreting) T cells in a quantitative manner and combines high sensitivity with relatively low T cell demand.     

应用支链DNA技术检测人乳头瘤病毒E6/E7mRNA在宫颈疾病筛查中的价值

目的评价支链DNA(bDNA)技术检测人乳头瘤病毒(HPV)E6/E7mRNA在宫颈疾病筛查中的临床价值。方法对262例宫颈疾病筛查妇女宫颈脱落细胞采用bDNA技术检测HPVE6/E7 mRNA、第二代杂交捕获技术(HC2)检测高危HPV,同时进行宫颈病理学检查。结果 (1)随着宫颈疾病级别升高,bDNA技术和HC2法检测HPV阳性率均呈现趋势性增加(χ2=65.397,P<0.001;χ2=65.018,P<0.001);(2)bDNA技术对总体人群检出阳性率为39.7%,HC2法检出阳性率为56.1%,HC2法显著高于bDNA技术(χ2=13.489,P<0.001),二者一致性一般(Kappa=0.501);(3)bDNA技术对CIN2+诊断的灵敏度为63.4%(95%CI:54.5%～72.3%),显著低于HC2法78.6%(95%CI:71.0%～86.2%)(χ2=5.549,P=0.018);而特异性为78.0%(95%CI:71.4%～84.6%),高于HC2的60.7%(95%CI:52.8%～68.5%)(χ2=9.798,P=0.002)。结论 HC2法检测高危HPV仍是目前宫颈疾病筛查...     

人乳头瘤病毒16型L1基因真核表达载体的构建及表达

目的从感染人乳头瘤病毒（HPV）的新鲜宫颈癌组织中扩增HPV16型晚期蛋白L1基因全长，构建表达载体，并验证目的蛋白的表达。方法根据基因全长设计一对特异引物，用PCR的方法从宫颈癌组织DNA，中获得L1基因，以pMD18T为克隆载体，构建重组质粒进行亚克隆，再以pcDNA3．1（＋）为载体构建表达质粒，转染至人肝细胞系H7702，提取蛋白，采用Western Blot方法检测HPV16-L1蛋白的表达。结果从长春地区宫颈癌临床标本克隆到的HPV16型L1蛋白编码序列与Genebank报道序列高度同源，其真核表达产物与特异性抗体能够特异性结合。结论成功构建重组表达质粒pcDNA3．1（＋）-HPV16-L1，为进一步研制HPV预防性疫苗创造基础条件。