Ya Chea Tsai

Generation and Characterization of DNA Vaccines Targeting the Nucleocapsid Protein of Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT Severe acute respiratory syndrome (SARS) is a serious threat to public health and the economy on a global scale. The SARS coronavirus (SARS-CoV) has been identified as the etiological agent for SARS. Thus, vaccination against SARS-CoV may represent an effective approach to controlling SARS. DNA vaccines are an attractive approach for SARS vaccine development, as they offer many advantages over conventional vaccines, including stability, simplicity, and safety. Our investigators have previously shown that DNA vaccination with antigen linked to calreticulin (CRT) dramatically enhances major histocompatibility complex class I presentation of linked antigen to CD8+ T cells. In this study, we have employed this CRT-based enhancement strategy to create effective DNA vaccines using SARS-CoV nucleocapsid (N) protein as a target antigen. Vaccination with naked CRT/N DNA generated the most potent N-specific humoral and T-cell-mediated immune responses in vaccinated C57BL/6 mice among all of the DNA constructs tested. Furthermore, mice vaccinated with CRT/N DNA were capable of significantly reducing the titer of challenging vaccinia virus expressing the N protein of the SARS virus. These results show that a DNA vaccine encoding CRT linked to a SARS-CoV antigen is capable of generating strong N-specific humoral and cellular immunity and may potentially be useful for control of infection with SARS-CoV.

Development of a DNA Vaccine Targeting Human Papillomavirus Type 16 Oncoprotein E6

ABSTRACT 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-2Kb, 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.

Ectopic Expression of Vascular Cell Adhesion Molecule-1 as a New Mechanism for Tumor Immune Evasion

Immune escape is an important reason why the immune system cannot control tumor growth, but how escape variants emerge during immunotherapy remains poorly understood. Here, we identify a new mechanism of tumor immune escape using an in vivo selection strategy. We generated a highly immune-resistant cancer cell line (P3) by subjecting a susceptible cancer cell line (P0/TC-1) to multiple rounds of in vivo immune selection. Microarray analysis of P0 and P3 revealed that vascular cell adhesion molecule-1 (VCAM-1) is up-regulated in the P3-resistant variant. Retroviral transfer of VCAM-1 into P0 significantly increased its resistance against a vaccine-induced immune response. Analysis of tumors showed a dramatic decrease in the number of tumor-infiltrating cluster of differentiation 8(+) (CD8(+)) T cells in the tumors expressing VCAM-1. In vitro transwell migration assays showed that VCAM-1 can promote the migration of CD8(+) T cells through its interaction with the alpha(4)beta(1) integrin. Site-directed mutagenesis of VCAM-1 at amino acid residues required for interaction with alpha(4)beta(1) integrin completely abolished the immune resistance conferred by VCAM-1 in vivo. Surface staining showed that most renal cell carcinomas (RCC) express VCAM-1, whereas an RCC that responded to vaccination was VCAM-1 negative. These data provide evidence that tumor expression of VCAM-1 represents a new mechanism of immune evasion and has important implications for the development of immunotherapy for human RCC.

Enhancing the therapeutic effect against ovarian cancer through a combination of viral oncolysis and antigen-specific immunotherapy.

Cancer therapy using oncolytic viruses represents a promising new approach for controlling ovarian cancer. In this study, we have circumvented the limitation of repeated vaccination by employing different virus vectors, Semliki Forest Virus (SFV) and vaccinia virus (VV) for boosting the immune response. We found that infection of tumor-bearing mice with VV followed by infection with SFV or vice versa leads to enhanced antitumor effects against murine ovarian surface epithelial carcinoma (MOSEC) tumors. Furthermore, infection with VV-ovalbumin (OVA) followed by infection with SFV-OVA or vice versa was found to lead to enhanced OVA-specific CD8(+) T-cell immune responses. In addition, we found that infection with SFV-OVA followed by infection with VV-OVA leads to enhanced antitumor effects in vivo and enhanced tumor killing in vitro through a combination of viral oncolysis and antigen-specific immunity. The clinical implications of this study are discussed.

Carrageenan as an adjuvant to enhance peptide-based vaccine potency.

New innovative therapies are urgently required in order to combat the high mortality and morbidity associated with advanced cancers. Antigen-specific cancer immunotherapy using peptide-based vaccination has emerged as an attractive approach for the control of cancers due to its simplicity and easy preparation. However, such an approach requires the employment of suitable adjuvants. In the current study, we explored the employment of a sulfated polysaccharide compound from red algae, carrageenan (CGN) as an adjuvant for their ability to generate antigen-specific immune responses and antitumor effects in mice vaccinated with human papillomavirus type 16 (HPV-16) E7 peptide vaccine. We found that carrageenan can significantly enhance the E7-specific immune responses generated by E7 peptide vaccination via the TLR4 activation pathway. In addition, carrageenan could enhance the protective and therapeutic antitumor effects generated by E7 peptide vaccination against E7-expressing tumors. Furthermore, the observed enhancement was not restricted to E7 antigen but was also applicable to other antigenic systems. We also found that other structurally similar compounds to CGN, such as dextran, also generated similar immune enhancement. Thus, our data suggest that CGN and its structurally related compounds may serve as innovative adjuvants for enhancing peptide-based vaccine potency.

Toll-like Receptor Agonist Imiquimod Facilitates Antigen-Specific CD8+ T-cell Accumulation in the Genital Tract Leading to Tumor Control through IFNγ

Purpose: Imiquimod is a Toll-like receptor 7 agonist used topically to treat external genital warts and basal cell carcinoma. We examined the combination of topical imiquimod with intramuscular administration of CRT/E7, a therapeutic human papillomavirus (HPV) vaccine comprised of a naked DNA vector expressing calreticulin fused to HPV16 E7. Experimental Design: Using an orthotopic HPV16 E6/E7+ syngeneic tumor, TC-1, as a model of high-grade cervical/vaginal/vulvar intraepithelial neoplasia, we assessed if combining CRT/E7 vaccination with cervicovaginal deposition of imiquimod could result in synergistic activities promoting immune-mediated tumor clearance. Results: Imiquimod induced cervicovaginal accumulation of activated E7-specific CD8+ T cells elicited by CRT/E7 vaccination. Recruitment was not dependent upon the specificity of the activated CD8+ T cells, but was significantly reduced in mice lacking the IFNγ receptor. Intravaginal imiquimod deposition induced upregulation of CXCL9 and CXCL10 mRNA expression in the genital tract, which are produced in response to IFNγ receptor signaling and attract cells expressing their ligand, CXCR3. The T cells attracted by imiquimod to the cervicovaginal tract expressed CXCR3 as well as CD49a, an integrin involved in homing and retention of CD8+ T cells at mucosal sites. Our results indicate that intramuscular CRT/E7 vaccination in conjunction with intravaginal imiquimod deposition recruits antigen-specific CXCR3+ CD8+ T cells to the genital tract. Conclusions: Several therapeutic HPV vaccination clinical trials using a spectrum of DNA vaccines, including vaccination in concert with cervical imiquimod, are ongoing. Our study identifies a mechanism by which these strategies could provide therapeutic benefit. Our findings support accumulating evidence that manipulation of the tumor microenvironment can enhance the therapeutic efficacy of strategies that induce tumor-specific T cells. Clin Cancer Res; 20(21); 5456–67. ©2014 AACR.

Xenogeneic Human p53 DNA Vaccination by Electroporation Breaks Immune Tolerance to Control Murine Tumors Expressing Mouse p53

The pivotal role of p53 as a tumor suppressor protein is illustrated by the fact that this protein is found mutated in more than 50% of human cancers. In most cases, mutations in p53 greatly increase the otherwise short half-life of this protein in normal tissue and cause it to accumulate in the cytoplasm of tumors. The overexpression of mutated p53 in tumor cells makes p53 a potentially desirable target for the development of cancer immunotherapy. However, p53 protein represents an endogenous tumor-associated antigen (TAA). Immunization against a self-antigen is challenging because an antigen-specific immune response likely generates only low affinity antigen-specific CD8+ T-cells. This represents a bottleneck of tumor immunotherapy when targeting endogenous TAAs expressed by tumors. The objective of the current study is to develop a safe cancer immunotherapy using a naked DNA vaccine. The vaccine employs a xenogeneic p53 gene to break immune tolerance resulting in a potent therapeutic antitumor effect against tumors expressing mutated p53. Our study assessed the therapeutic antitumor effect after immunization with DNA encoding human p53 (hp53) or mouse p53 (mp53). Mice immunized with xenogeneic full length hp53 DNA plasmid intramuscularly followed by electroporation were protected against challenge with murine colon cancer MC38 while those immunized with mp53 DNA were not. In a therapeutic model, established MC38 tumors were also well controlled by treatment with hp53 DNA therapy in tumor bearing mice compared to mp53 DNA. Mice vaccinated with hp53 DNA plasmid also exhibited an increase in mp53-specific CD8+ T-cell precursors compared to vaccination with mp53 DNA. Antibody depletion experiments also demonstrated that CD8+ T-cells play crucial roles in the antitumor effects. This study showed intramuscular vaccination with xenogeneic p53 DNA vaccine followed by electroporation is capable of inducing potent antitumor effects against tumors expressing mutated p53 through CD8+ T cells.

Cancer Immunotherapy Using Irradiated Tumor Cells Secreting Heat Shock Protein 70

Ovarian cancer is responsible for the highest mortality rate among patients with gynecologic malignancies. Therefore, there is an emerging need for innovative therapies for the control of advanced ovarian cancer. Immunotherapy has emerged as a potentially plausible approach for the control of ovarian cancer. In the current study, we have generated heat shock protein 70 (Hsp70)-secreting murine ovarian cancer cells that express luciferase (MOSEC/luc). Hsp70 has been shown to target and concentrate antigenic peptides in dendritic cells and is also able to activate dendritic cells. We characterized the antigen-specific immune response and the antitumor effect of the MOSEC/luc cells expressing Hsp70 using noninvasive luminescence images to measure the amount of ovarian tumors in the peritoneal cavity of mice. We found that mice challenged with MOSEC/luc cells expressing Hsp70 generate significant antigen-specific CD8+ T-cell immune responses. Furthermore, we also found that mice vaccinated with irradiated MOSEC/luc cells expressing Hsp70 generate significant therapeutic effect against MOSEC/luc cells. In addition, we have shown that CD8+, natural killer, and CD4+ cells are important for protective antitumor effect generated by irradiated tumor cell-based vaccines expressing Hsp70. Moreover, we also found that CD40 receptor is most important, followed by Toll-like receptor 4 receptor, for inhibiting in vivo tumor growth of the viable MOSEC/luc expressing Hsp70. Thus, the use of Hsp70-secreting ovarian tumor cells represents a potentially effective therapy for the control of lethal ovarian cancer.

Creation of a Merkel cell polyomavirus small T antigen-expressing murine tumor model and a DNA vaccine targeting small T antigen.

BackgroundMerkel cell polyomavirus (MCPyV) is a DNA virus expressing transcripts similar to the large T (LT) and small T (ST) transcripts of SV40, which has been implicated in the pathogenesis of Merkel cell carcinoma (MCC), a rare and highly aggressive neuroendocrine skin cancer. MCPyV LT antigen expression was found to be a requirement for MCC tumor maintenance and ST protein also likely contributes to the carcinogenesis of MCC. Previously, we have identified the probable immunodominant epitope of MCPyV LT and developed a DNA vaccine encoding this epitope linked to calreticulin. The LT-targeting DNA vaccine generated prolonged survival, decreased tumor size and increased LT-specific CD8+ T cells in tumor-bearing mice.ResultsIn this study, we developed a MCPyV ST-expressing tumor cell line from B16 mouse melanoma cells. We then utilized this ST-expressing tumor cell line to test the efficacy of a DNA vaccine encoding ST. In ST-expressing tumor-bearing mice, this vaccine, pcDNA3-MCC/ST, generated a significant number of ST antigenic peptide-specific CD8+ T cells and experienced markedly enhanced survival compared to mice vaccinated with empty vector.ConclusionsThe formation of an effective vaccine against MCPyV has the potential to advance the field of MCC therapy and may contribute to the control of this severe malignancy through immunotherapy. Both of the innovative technologies presented here provide opportunities to develop and test MCPyV-targeted therapies for the control of Merkel cell carcinoma.

Tumor-Targeted Delivery of IL-2 by NKG2D Leads to Accumulation of Antigen-Specific CD8+ T Cells in the Tumor Loci and Enhanced Anti-Tumor Effects

Interleukin-2 (IL-2) has been shown to promote tumor-specific T-cell proliferation and differentiation but systemic administration of IL-2 results in significant toxicity. Therefore, a strategy that can specifically deliver IL-2 to the tumor location may alleviate concerns of toxicity. Because NKG2D ligands have been shown to be highly expressed in many cancer cells but not in healthy cells, we reason that a chimeric protein consisting of NKG2D linked to IL-2 will lead to the specific targeting of IL-2 to the tumor location. Therefore, we created chimeric proteins consisting of NKG2D linked to Gaussia luciferase (GLuc; a marker protein) or IL-2 to form NKG2D-Fc-GLuc and NKG2D-Fc-IL2, respectively. We demonstrated that NKG2D linked to GLuc was able to deliver GLuc to the tumor location in vivo. Furthermore, we showed that TC-1 tumor-bearing mice intramuscularly injected with DNA encoding NKG2D-Fc-IL2, followed by electroporation, exhibited an increased number of luciferase-expressing E7-specific CD8+ T cells at the tumor location. More importantly, treatment with the DNA construct encoding NKG2D-Fc-IL2 significantly enhanced the therapeutic anti-tumor effects generated by intradermal vaccination with therapeutic HPV DNA in tumor-bearing mice. Therefore, by linking NKG2D to IL2, we are able to specifically deliver IL-2 to the tumor location, enhancing antigen-specific T-cell immune response and controlling tumor growth. Our approach represents a platform technology to specifically deliver proteins of interest to tumor loci.