[show abstract][hide abstract] ABSTRACT: We have examined non-replicative human papillomavirus (HPV) pseudovirions as an approach in the delivery of naked DNA vaccines without safety concerns associated with live viral vectors. In this study, we have generated HPV-16 pseudovirions encapsidating a DNA vaccine encoding the model antigen, ovalbumin (OVA) (HPV16-OVA pseudovirions). Vaccination with HPV16-OVA pseudovirions subcutaneously elicited significantly stronger OVA-specific CD8+ T-cell immune responses compared with OVA DNA vaccination via gene gun in a dose-dependent manner. We showed that a single amino acid mutation in the L2 minor capsid protein that eliminates the infectivity of HPV16-OVA pseudovirion significantly decreased the antigen-specific CD8+ T-cell responses in vaccinated mice. Furthermore, a subset of CD11c+ cells and B220+ cells in draining lymph nodes became labeled on vaccination with fluorescein isothiocyanate-labeled HPV16-OVA pseudovirions in injected mice. HPV pseudovirions were found to infect bone marrow-derived dendritic cells (BMDCs) in vitro. We also showed that pretreatment of HPV16-GFP pseudovirions with furin leads to enhanced HPV16-OVA pseudovirion infection of BMDCs and OVA antigen presentation. Our data suggest that DNA vaccines delivered using HPV pseudovirions represent an efficient delivery system that can potentially affect the field of DNA vaccine delivery.
[show abstract][hide abstract] ABSTRACT: Human papillomavirus (HPV), particularly type 16, has been associated with a subset of head and neck cancers. The viral-encoded oncogenic proteins E6 and E7 represent ideal targets for immunotherapy against HPV-associated head and neck cancers. DNA vaccines have emerged as attractive approaches for immunotherapy due to its simplicity, safety and ease of preparation. Intradermal administration of DNA vaccine by means of gene gun represents an efficient method to deliver DNA directly into dendritic cells for priming antigen-specific T cells. We have previously shown that a DNA vaccine encoding an invariant chain (Ii), in which the class II-associated Ii peptide (CLIP) region has been replaced by a Pan-DR-epitope (PADRE) sequence to form Ii-PADRE, is capable of generating PADRE-specific CD4+ T cells in vaccinated mice. In the current study, we hypothesize that a DNA vaccine encoding Ii-PADRE linked to E6 (Ii-PADRE-E6) will further enhance E6-specific CD8+ T cell immune responses through PADRE-specific CD4+ T-helper cells. We found that mice vaccinated with Ii-PADRE-E6 DNA generated comparable levels of PADRE-specific CD4+ T-cell immune responses, as well as significantly stronger E6-specific CD8+ T-cell immune responses and antitumor effects against the lethal challenge of E6-expressing tumor compared with mice vaccinated with Ii-E6 DNA. Taken together, our data indicate that vaccination with Ii-E6 DNA with PADRE replacing the CLIP region is capable of enhancing the E6-specific CD8+ T-cell immune response generated by the Ii-E6 DNA. Thus, Ii-PADRE-E6 represents a novel DNA vaccine for the treatment of HPV-associated head and neck cancer and other HPV-associated malignancies.
[show abstract][hide abstract] ABSTRACT: Intradermal administration of DNA vaccines via a gene gun represents a feasible strategy to deliver DNA directly into the professional antigen-presenting cells (APCs) in the skin. This helps to facilitate the enhancement of DNA vaccine potency via strategies that modify the properties of APCs. We have previously demonstrated that DNA vaccines encoding human papillomavirus type 16 (HPV-16) E7 antigen linked to calreticulin (CRT) are capable of enhancing the E7-specific CD+ T-cell immune responses and antitumor effects against E7-expressing tumors. It has also been shown that cluster (short-interval) DNA vaccination regimen generates potent immune responses in a minimal time frame. Thus, in the current study we hypothesize that the cluster intradermal CRT/E7 DNA vaccination will generate significant antigen-specific CD8+ T-cell infiltrates in E7-expressing tumors in tumor-bearing mice, leading to an increase in apoptotic tumor cell death. We found that cluster intradermal CRT/E7 DNA vaccination is capable of rapidly generating a significant number of E7-specific CD8+ T cells, resulting in significant therapeutic antitumor effects in vaccinated mice. We also observed that cluster intradermal CRT/E7 DNA vaccination in the presence of tumor generates significantly higher E7-specific CD8+ T-cell immune responses in the systemic circulation as well as in the tumors. In addition, this vaccination regimen also led to significantly lower levels of CD4+Foxp3+ T-regulatory cells and myeloid suppressor cells compared to vaccination with CRT DNA in peripheral blood and in tumor-infiltrating lymphocytes, resulting in an increase in apoptotic tumor cell death. Thus, our study has significant potential for future clinical translation.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The potency of DNA vaccines may be affected by the efficiency of intracellular processing and MHC class I presentation of encoded antigens. Since a single-chain trimer (SCT) composed of peptide, beta2-microglobulin (beta2m), and MHC class I heavy chain has been shown to bypass antigen processing and lead to stable presentation of peptides, we investigated the efficacy of a DNA vaccine encoding a SCT composed of an immunodominant CTL epitope of human papillomavirus type 16 (HPV-16) E6 antigen, beta2m, and H-2Kb MHC class I heavy chain (pIRES-E6-beta2m-Kb). Transfection of 293 cells with pIRES-E6-beta2m-Kb can bypass antigen processing and lead to stable presentation of E6 peptide. Furthermore, C57BL/6 mice vaccinated with pIRES-E6-beta2m-Kb exhibited significantly increased E6 peptide-specific CD8+ T-cell immune responses compared to mice vaccinated with DNA encoding wild-type E6. Most importantly, 100% of mice vaccinated with pIRES-E6-beta2m-Kb DNA were protected against a lethal challenge of E6-expressing TC-1 tumor cells. In contrast, all mice vaccinated with wild-type E6 DNA or control plasmid DNA grew tumors. Our data indicate that a DNA vaccine encoding a SCT can lead to stable enhanced MHC class I presentation of encoded antigenic peptide and may be useful for improving DNA vaccine potency to control tumors or infectious diseases.