Activation of dendritic cells and induction of T cell responses by HPV 16 L1/E7 chimeric virus-like particles are enhanced by CpG ODN or sorbitol

Chimeric human papillomavirus-like particles, consisting of human papillomavirus (HPV) 16 L1-E7 fusion proteins [HPV 16 L1/E7 chimeric virus-like particles (CVLP)], are a vaccine candidate for treatment and prevention of cervical cancer. Although in preclinical studies CVLPs were shown to induce neutralizing antibodies and L1- and E7-specific T cell responses, the results of a recent clinical trial emphasized the need of improved immunogenicity of CVLPs. Here we studied the interaction of HPV 16 L1/E7 CVLPs with mouse bone marrow-derived dendritic cells (BMDCs) activated with different immune adjuvants. We found that lipopolysaccharides (LPS), unmethylated CpG motifs (CpG ODN) and sorbitol enhanced CVLP-induced stimulation of C57BL/6 mouse BMDCs as revealed by increased levels of CD40, CD80, MHC II and CD54 at the cell surface. CpG ODN and sorbitol also enhanced the presentation of Db-restricted cytotoxic T lymphocyte epitopes to HPV 16 L1- or E7-specific T lymphocytes after loading of CVLPs onto BMDCs. Treatment of BMDCs with CpG ODN in combination with CVLPs improved in vitro priming of naive T lymphocytes by CVLP-loaded BMDCs. In vivo, CVLP-loaded BMDCs were more immunogenic as compared with injection of CVLPs alone. CpG ODN and sorbitol further enhanced priming of antigen-specific T cell responses. Our data demonstrate that CpG ODN- or sorbitol-activated BMDCs substantially increase the immunogenicity of CVLPs. Implementing our results in clinical trial protocols may lead to improved activity of therapeutic HPV vaccines for the treatment of HPV-induced cancer.     

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.     

Evaluation of HBs, HBc, and frCP virus-like particles for expression of human papillomavirus 16 E7 oncoprotein epitopes

OBJECTIVES: In an attempt to develop virus-like particles (VLPs) as experimental vaccine against human papilloma virus (HPV)-induced tumours, the HPV16 E7 oncoprotein epitopes spanning amino acid (aa) residues 35-98 were expressed on three proteins capable of VLP formation: hepatitis B virus (HBV) surface (HBs) and core (HBc) antigens, and RNA phage fr coats (frCP). METHODS: The profile of immunoglobulin isotypes induced in Balb/C mice after immunization with purified chimeric proteins was studied. RESULTS: The HBs*-E7(35-54) protein expressing E7 residues 35-54 between residues 139 and 142 of the HBs carrier formed HBs-like particles in Saccharomyces cerevisiae. The HBc Delta-E7(35-98), but not the frCP-E7(35-98), ensured VLP formation in Escherichia coli. In Balb/C mice, the HBs*-E7(35-54) VLPs predominantly induced an anti-E7 antibody, but not anti-HBs carrier response, whereas the HBc Delta-E7(35-98) VLPs induced a lower anti-E7 compared to anti-HBc carrier response. The frCP-E7(35-98) protein elicited equally high antibody responses to both E7 and frCP carrier. Analysis of the immunoglobulin G isotype profile of the antibodies induced by the E7-carrying chimeras showed that the HBs and frCP derivatives were capable of eliciting the Th1 and Th2 subsets of T helper cells, whereas the HBc-derived chimeras elicited only the Th2 subset. CONCLUSIONS: The HBs and HBc, but not frCP carriers support an efficient outcome for VLPs carrying the HPV16 E7 epitopes. All chimeric proteins may be regarded as potential vaccine candidates.     

A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E

We have reported the identification of human gene MAGE-1, which directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes (CTL). We show here that CTL directed against this antigen, which was named MZ2-E, recognize a nonapeptide encoded by the third exon of gene MAGE-1. The CTL also recognize this peptide when it is presented by mouse cells transfected with an HLA-A1 gene, confirming the association of antigen MZ2-E with the HLA-A1 molecule. Other members of the MAGE gene family do not code for the same peptide, suggesting that only MAGE-1 produces the antigen recognized by the anti-MZ2-E CTL. Our results open the possibility of immunizing HLA-A1 patients whose tumor expresses MAGE-1 either with the antigenic peptide or with autologous antigen-presenting cells pulsed with the peptide.     

Genetic immunization against cervical carcinoma: induction of cytotoxic T lymphocyte activity with a recombinant alphavirus vector expressing human papillomavirus type 16 E6 and E7

Infection of genital epithelial cells with human papillomavirus (HPV) types 16 and 18 is closely associated with the development of cervical carcinoma. The transforming potential of these high-risk HPVs depends on the expression of the E6 and E7 early viral gene products. Since the expression of E6 and E7 is selectively maintained in premalignant and malignant cervical lesions these proteins are attractive candidates for immunotherapeutic and prophylactic strategies. This report describes the construction, characterization and the in vivo immunotherapeutic potential of recombinant Semliki Forest virus (SFV) expressing the HPV16 E6 and E7 proteins (SFV-E6E7). Western blot analysis and immunofluorescence staining demonstrated expression of E6 and E7 in BHK cells infected with SFV-E6E7. Immunization of mice with SFV-E6E7 resulted in an efficient in vivo priming of HPV-specific CTL activity. The induced CTL lysed murine tumor cells transformed with the HPV16 genome and EL4 cells loaded with an immunodominant class I-binding HPV E7 peptide. CTLs could reproducibly be induced by immunization with three injections of as few as 10(5) infectious units of SFV-E6E7. Protection from tumor challenge was studied using the tumor cell line TC-1. Immunization with 5 x 10(6) SFV-E6E7 particles protected 40% of the mice from tumor challenge. These results indicate that E6E7 expression by the efficient and safe recombinant SFV system represents a promising strategy for immunotherapy or immunoprophylaxis of cervical carcinoma.     

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.     

Characterization of HLA-A2-restricted HPV-16 E7-specific CD8(+) T-cell immune responses induced by DNA vaccines in HLA-A2 transgenic mice

We have recently demonstrated that linkage of DNA-encoding calreticulin to DNA-encoding human papillomavirus-16 E7 antigen strongly enhances the efficacy of DNA vaccines against E7-expressing tumors in animal models. In this study, as a prelude to clinical translation, we characterized the ability of DNA-encoding calreticulin linked to DNA-encoding E7 antigen to generate HLA-A2-restricted E7-specific CD8(+) T-cell responses in HLA-A2 (AAD) transgenic mice, as well as antitumor effects against an E7(+) HLA-A2(+) tumor cell line, TC-1/A2. Our results show that while vaccination with CRT/E7 DNA generates strong H-2D(b)-restricted E7 (amino acid (aa)49-57)-specific CD8(+) T-cell immune responses in both C57BL/6 and HLA-A2 (AAD) transgenic mice, no such responses were generated to HLA-A2-restricted epitopes in either type of mouse. In contrast, vaccination with DNA-encoding calreticulin linked to DNA encoding a mutant version of E7 with a deleted aa49-57 epitope leads to the generation of an HLA-A2-restricted E7 (aa11-20)-specific CTL response in HLA-A2 (AAD) transgenic mice. More importantly, vaccination with CRT/mtE7 (del aa49-57) DNA protects against a lethal challenge with TC-1/A2 tumor cells in HLA-A2 (AAD) transgenic mice. Furthermore, our in vitro studies demonstrate that the presence of the E7 (aa49-57) epitope does not suppress presentation of the HLA-A2-restricted E7 (aa11-20) epitope through MHC class I molecules. Thus, the predominant E7 aa49-57-specific CD8+ T-cell immune response in HLA-A2 transgenic mice vaccinated with CRT/E7 is likely due to preferred expansion of E7 aa49-57-specific CD8(+) T cells in vaccinated mice. These results highlight the importance of epitope immunodominance in the evaluation of immune responses in HLA-A2 (AAD) transgenic mice.     

Cancer immunotherapy using Sindbis virus replicon particles encoding a VP22-antigen fusion

Alphavirus vectors have emerged as a strategy for the development of cancer vaccines and gene therapy applications. The availability of a new packaging cell line (PCL), which is capable of generating alphavirus replicon particles without contamination from replication-competent virus, has advanced the field of vaccine development. This replication-defective vaccine vector has potential advantages over naked nucleic acid vaccines, such as increased efficiency of gene delivery and large-scale production. We have developed a new strategy to enhance nucleic acid vaccine potency by linking VP22, a herpes simplex virus type 1 (HSV-1) tegument protein, to a model antigen. This strategy facilitated the spread of linked E7 antigen to neighboring cells. In this study, we created a recombinant Sindbis virus (SIN)-based replicon particle encoding VP22 linked to a model tumor antigen, human papillomavirus type 16 (HPV-16) E7, using a stable SIN PCL. The linkage of VP22 to E7 in these SIN replicon particles resulted in a significant increase in the number of E7-specific CD8(+) T cell precursors and a strong antitumor effect against E7-expressing tumors in vaccinated C57BL/6 mice relative to wild-type E7 SIN replicon particles. Furthermore, a head-to-head comparison of VP22-E7-containing naked DNA, naked RNA replicons, or RNA replicon particle vaccines indicated that SINrep5-VP22/E7 replicon particles generated the most potent therapeutic antitumor effect. Our results indicated that the VP22 strategy used in the context of SIN replicon particles may facilitate the generation of a highly effective vaccine for widespread immunization.     

A virosomal immunization strategy against cervical cancer and pre-malignant cervical disease

In this study, we demonstrate that fusion-active virosomes, containing recombinant human papillomavirus type 16 (HPV16) E7 protein antigen, are capable of inducing a robust class I MHC-restricted cytotoxic T-lymphocyte (CTL) response against HPV-transformed tumour cells in a murine model system. Virosomes are reconstituted viral envelopes, which do not contain the genetic material of the native virus. During the reconstitution process, protein antigens can be encapsulated within the virosomes. In the present study, we used virosomes derived from influenza virus. These virosomes retain the cell binding and membrane fusion characteristics of native influenza virus, and have the capacity to deliver encapsulated antigens to the cytosol of antigen-presenting cells through fusion from within acidic endosomes. After immunization of mice with virosomes containing encapsulated HPV16 E7 protein, the animals developed a strong E7-specific CTL response as assessed by 51Cr release measurements and MHC tetramer staining of spleen cells. Immunization with E7-containing virosomes also resulted in E7-specific antibody responses. In tumour challenge experiments, immunization of mice with E7-containing virosomes prevented tumour outgrowth in >70% of the animals. Thus, influenza-derived virosomes with encapsulated HPV E7 protein antigen act as an excellent vaccine delivery system for induction of cellular immunity against HPV-transformed cells and represent a promising immunotherapeutic vaccine for the treatment of (precursor lesions of) cervical cancer.     

Immunizations with chimeric hepatitis B virus-like particles to induce potential anti-hepatitis C virus neutralizing antibodies

BACKGROUND: Virus-like particles (VLPs) are highly immunogenic and proven to induce protective immunity. The small surface antigen (HBsAg-S) of hepatitis B virus (HBV) self-assembles into VLPs and its use as a vaccine results in protective antiviral immunity against HBV infections. Chimeric HBsAg-S proteins carrying foreign epitopes allow particle formation and have the ability to induce anti-foreign humoral and cellular immune responses. METHODS/RESULTS: The insertion of the hypervariable region 1 (HVR1) sequence derived from the envelope protein 2 (E2) of hepatitis C virus (HCV) into the major antigenic site of HBsAg-S ('a'-determinant) resulted in the formation of highly immunogenic VLPs that retained the antigenicity of the inserted HVR1 sequence. BALB/c mice were immunized with chimeric VLPs, which resulted in antisera with anti-HCV activity. The antisera were able to immunoprecipitate native HCV envelope complexes (E1E2) containing homologous or heterologous HVR1 sequences. HCV E1E2 pseudotyped HIV-1 particles (HCVpp) were used to measure entry into HuH-7 target cells in the presence or absence of antisera that were raised against chimeric VLPs. Anti-HVR1 VLP sera interfered with entry of entry-competent HCVpps containing either homologous or heterologous HVR1 sequences. Also, immunizations with chimeric VLPs induced antisurface antigen (HBsAg) antibodies, indicating that HBV-specific antigenicity and immunogenicity of the 'a'-determinant region is retained. CONCLUSIONS: A multivalent vaccine against different pathogens based on the HBsAg delivery platform should be possible. We hypothesize that custom design of VLPs with an appropriate set of HCV-neutralizing epitopes will induce antibodies that would serve to decrease the viral load at the initial infecting inoculum.     

Enhanced immunogenicity of human papillomavirus 16 L1 genetic vaccines fused to an ER-targeting secretory signal peptide and RANTES

To increase the potency of human papillomavirus (HPV) DNA vaccines, we constructed a series of HPV16 L1 vaccines genetically fused with a secretion signal and/or immune cell-recruiting RANTES. The DNA vaccines encoding secretory HPV L1 were constructed by inserting HPV L1 gene into a vector with an ER-targeting secretory signal sequence. The expression plasmid encoding secretory HPV L1 (pER/L1) was fused with cDNA of RANTES, generating pER/L1/R. For comparison, HPV L1 genes were cloned into pVAX1 vector with no signal sequence (pL1), and further linked to the N-terminus (pL1/R) or C-terminus of RANTES (pR/L1). The secretion of L1 proteins was observed in the pER/L1, pER/L1/R, and pR/L1-transfected cells, except the pL1/R-transfected group. Cytoplasmic localization of L1 protein was observed in the cells transfected with pL1/R, but not with pER/L1/R at 48 h after transfection. In mice, RANTES-fused vaccines more effectively elicited the levels of HPV16 L1-specific IgG and IgG2a antibodies than pL1. Of RANTES-fused vaccines, pER/L1/R encoding the secreted fusion protein induced the highest humoral and CD8(+) T-cell-stimulating responses. These results suggest that the immunogenicity of HPV L1 DNA vaccines could be enhanced by genetic fusion to a chemokine and secretory signal peptide sequences.     

Dendritic cell immunization induces Nonprotective WT1-specific CTL responses in mouse

In the present article we describe the immunogenicity in the mouse of 2 epitopes from the tumor-associated antigen Wilms tumor 1 antigen (WT1). The newly described K-restricted pWT330 epitope stimulates high-avidity allo-major histocompatibility complex restricted cytotoxic T lymphocyte (CTL) capable of killing WT1-expressing tumor cell lines. The epitope pWT126 has been previously described as a D-restricted CTL epitope. Both epitopes are weakly immunogenic as immunization with incomplete Freund adjuvant induced poor CTL responses. In contrast, when coated onto dendritic cells (DCs) both peptides readily induced CTL responses. However, these peptide-specific CTL were of low avidity and unable to recognize WT1-expressing tumor cells in vitro and to protect against tumor challenge in vivo. In contrast, vaccination with DCs coated with peptides derived from the nonself antigen ovalbumin (OVA) induced CTL that recognized OVA-expressing tumor cells and protected against tumor growth in vivo. These data show that although DC vaccination readily stimulated CTL against WT1 peptides, these CTL did not display antitumor activity in vitro and in vivo. This suggests that tolerance to the 2 WT1 epitopes interferes with the generation of protective CTL immunity in mice.     

HPV L1壳蛋白在宫颈病变中的表达及与HR-HPV DNA的关系

目的研究人乳头瘤病毒(HPV)L1壳蛋白在宫颈病变中的表达及与高危型人乳头瘤病毒(HR-HPV)DNA的关系,为宫颈病变的早期诊断提供预测指标,为HPV疫苗的研制提供理论依据。方法采用免疫细胞化学方法和第二代杂交捕获法(HC-Ⅱ),分别检测272例女性HPV L1壳蛋白的表达和高危型HPV(HR-HPV)病毒载量,组织活检复查宫颈病变情况。结果 HR-HPV DNA感染者中,随着宫颈病变级别(CINⅠ及以上)的升高,HPV L1壳蛋白阳性表达率增高(x~2=35.176,P0.001),HPV L1壳蛋白诊断CINⅡ及以上的灵敏度为89.77%,特异度为45.12%;按年龄分层后,HPV L1壳蛋白表达与宫颈病变的OR均小于1;小于35岁研究对象中,病毒载量与HPV L1壳蛋白阳性表达率之间存在剂量反应关系(x~2=12.915,P0.001)。结论 HPV L1壳蛋白的缺失与宫颈癌及癌前病变的发生、发展密切相关,年轻女性HPV L1壳蛋白的表达与HPVDNA载量存在剂量反应关系。     

HPV58型衣壳蛋白L1和L2基因的克隆表达及病毒样颗粒的装配

目的大量表达和纯化HPV58衣壳蛋白,形成病毒相似颗粒。方法制备携带HPV58L1和L2基因的重组杆状病毒,转染昆虫细胞,在真核细胞中大量表达HPV58L1和L2蛋白并进行纯化。电镜观察病毒相似颗粒的形成情况。免疫小鼠制备特异性抗血清。结果 HPV58L1和L2蛋白在真核细胞中被高效表达,电镜下见表达纯化的病毒衣壳蛋白L1与L2共同组装形成病毒相似颗粒(VLPs),用其免疫小鼠能产生针对HPV58L1蛋白的高滴度的特异性抗体。结论在真核细胞中大量表达HPV58L1和L2蛋白,纯化后能形成病毒相似颗粒,并具备很强的免疫原性,有望用其制作宫颈癌预防性疫苗。     

Papillomavirus virus-like particles as anticancer vaccines

Papillomavirus virus-like particles (VLPs) are empty, non-replicative, non-infectious particles that retain conformationally correct epitopes for the generation of antibody responses to the viral capsid proteins. Chimeric human papillomavirus (HPV) virus-like particles incorporating non-structural virus proteins offer an exciting approach for combined prophylactic and therapeutic vaccines against HPV-induced lesions. Both HPV VLPs and chimeric VLPs can induce potent humoral and cellular immune responses when injected into mice, leading to the generation of virus-neutralizing antibodies, priming of CD8+ T-cells and activation of cytotoxic T-cell effector functions. This review summarizes recent advances in the production of chimeric VLPs, the immune response elicited by VLPs and chimeric VLPs, and their ability to generate strong protective and therapeutic antitumor immune responses.     

Does B7-1 expression confer antigen-presenting cell capacity to tumors in vivo?

Tumors engineered to express the costimulatory molecule B7-1 can elicit CD8+ cytotoxic T lymphocyte (CTL)-dependent antitumor responses in immunocompetent mice. It has been postulated that this result reflects direct priming of CTL by the modified tumor in vivo. Previous studies of the immune response to a B7-1- murine colon carcinoma expressing influenza nucleoprotein (NP) as a model tumor antigen have demonstrated the crucial role of bone marrow-derived antigen-presenting cells (APCs) in the priming of NP-specific CTL in vivo. In this system, no evidence of direct CTL priming by tumor was detected. We have performed a similar analysis to determine if B7-1 transfectant of this tumor results in the direct priming of CTL, and to compare this response to that primed by host APCs. When H-2b-->H-2bxd bone marrow chimeras were immunized with a single injection of CT26/NP/B7-1 (H-2d), NP-specific CTL were detected that were restricted to the bone marrow haplotype (H- 2b), but not to the tumor haplotype. In contrast, CTL recognizing the NP antigenic epitope in the context of the tumor's major histocompatibility complex were detectable only after multiple immunizations. These results suggest that whereas B7-1+ tumor vaccines result in some degree of direct presentation to CD8+ T cells, the dominant mechanism of CTL priming is through the uptake and presentation of tumor antigens by bone marrow-deprived APCs. However, repeated immunization with B7-1+ tumor cells can efficiently expand the directly primed CD8+ CTL population.     

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.     

Gene gun-mediated DNA vaccination induces antitumor immunity against human papillomavirus type 16 E7-expressing murine tumor metastases in the liver and lungs

DNA vaccination has emerged as an attractive approach for tumor immunotherapy. The aim of this study was to evaluate the potency of DNA vaccines in preventing and treating the liver and lung metastases of a human papillomavirus-16 (HPV-16) E7-expressing murine tumor (TC-1). We used the gene gun method to vaccinate C57BL/6 mice intradermally with DNA vaccines containing the HPV-16 E7 gene, the E7 gene linked to the sorting signals of the lysosome-associated membrane protein-1 (Sig/E7/ LAMP-1), or the 'empty' plasmid vector. The in vivo antitumor immunity was analyzed in both tumor prevention and tumor regression experiments. In addition, cytotoxic T lymphocyte (CTL) assays, enzyme-linked immunospot assay and enzyme-linked immunoabsorbent assay were used to assess the E7-specific T cell-mediated and humoral immunity. Mice vaccinated with Sig/E7/LAMP-1 DNA generated the strongest E7-specific CTL activities, the highest numbers of E7-specific CD8+ cell precursors and the highest titers of E7-specific antibodies. While both E7 DNA and Sig/E7/LAMP-1 DNA generated potent antitumor immunity in the liver and lung metastases models, the Sig/E7/LAMP-1 DNA was more potent under stringent conditions. DNA vaccination with E7-expressing plasmids was effective in controlling liver and lung metastases of an E7-expressing murine tumor. Our data suggest that antigen-specific DNA vaccination can potentially be applied to control liver and lung metastases of tumors with defined tumor-specific antigens.     

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.     

Immune responses against human papillomavirus (HPV) infection and evasion of host defense in cervical cancer

Human papillomavirus (HPV) is the most important etiological factor for cervical cancer. A recent study demonstrated that more than 20 HPV types were thought to be oncogenic for uterine cervical cancer. Notably, more than one-half of women show cervical HPV infections soon after their sexual debut, and about 90 % of such infections are cleared within 3 years. Immunity against HPV might be important for elimination of the virus. The innate immune responses involving macrophages, natural killer cells, and natural killer T cells may play a role in the first line of defense against HPV infection. In the second line of defense, adaptive immunity via cytotoxic T lymphocytes (CTLs) targeting HPV16 E2 and E6 proteins appears to eliminate cells infected with HPV16. However, HPV can evade host immune responses. First, HPV does not kill host cells during viral replication and therefore neither presents viral antigen nor induces inflammation. HPV16 E6 and E7 proteins downregulate the expression of type-1 interferons (IFNs) in host cells. The lack of co-stimulatory signals by inflammatory cytokines including IFNs during antigen recognition may induce immune tolerance rather than the appropriate responses. Moreover, HPV16 E5 protein downregulates the expression of HLA-class 1, and it facilitates evasion of CTL attack. These mechanisms of immune evasion may eventually support the establishment of persistent HPV infection, leading to the induction of cervical cancer. Considering such immunological events, prophylactic HPV16 and 18 vaccine appears to be the best way to prevent cervical cancer in women who are immunized in adolescence.