While historically uncommon, merkel cell carcinoma (MCC) incidence has risen steadily over recent years[1, 2]. MCC is a cutaneous neoplasm that originates from the mechanoreceptor merkel cells, which are derived from the embryonic neural crest. Highly malignant, this disease is prone to metastasis, and therefore afflicted patients have unfavorable prognosis and high mortality rates. MCC is among the most aggressive of skin cancers with patient mortality greater than that of melanoma. Most often, MCC sufferers are seniors, immunosuppressed post-transplant patients, and immunodeficient individuals; with the disease first appearing at sun-exposed areas such as the head and neck.
Investigation into the contributing factors that lead to MCC development has determined a possible infectious origin for the disease, specifically, the merkel cell polyomavirus (MCPyV). Studies throughout the world have shown that approximately 80% of MCCs contain MCPyV DNA. It is unlikely that MCPyV is a passenger virus since tumor-derived MCPyV LT antigen contains unique mutations not found in wild-type virus. Furthermore, the viral genome is monocolonally integrated into the cell genome prior to tumor cell clonal expansion.
MCPyV is a small, oncogenic, virus with double-stranded circular DNA genome that is supercoiled. The viral genome contains a T antigen oncoprotein locus that can be expressed as four different alternatively spliced transcripts: the large T (LT) antigen, the two small T (ST) antigens, and the 57kT antigen. It is likely that MCPyV is the etiologic agent responsible for the majority of MCC development, although approximately 20% of MCCs are MCPyV-negative. Furthermore, MCPyV infection is ubiquitous whereas the development of MCCs is unusual[9, 10]. This has led to the speculation that MCPyV-positive MCCs entail specific conditions that enable viral-mediated oncogenesis.
The pathogenesis of MCPyV-positive MCC involves mechanisms that interfere with cell cycle checkpoints either through the disruption of regulatory sites during viral integration or through the inhibition of tumor suppressors. MCPyV DNA extracted from MCC cells has been found to contain mutations that cause the virus to be non-replicative. The LT antigen contains a premature stop codon that leads to the loss of helicase located at the C-terminus, which is necessary for viral replication. The abolishment of viral replication ensures the infected cell is safe from lytic cycle-induced apoptosis. Since the oncogenic N-terminus containing the retinoblastoma (RB) tumor suppressor protein-binding motif is preserved, the truncated LT antigen can instigate the transformation and proliferation of infected cells[11, 12]. The mutational prerequisites that permit MCPyV-induced MCC may explain the frequency of disease development on the areas of the skin that are constantly exposed to UV.
In a previous study, we demonstrated that a DNA vaccine encoding a truncated LT antigen was able to produce antitumor effects against LT-expressing tumor cells. The efficacy of the codon-optimized DNA vaccine was found to be predominantly a result of CD4+ T cells. However, we also determined that natural killer (NK) cells and CD8+ T cells have modest contributions to the therapeutic effects of the DNA vaccine. Consequently, we decided to examine if the DNA vaccine encoding LT antigen could be tailored to favor the generation of LT-specific CD8+ T cells. Tumor-infiltrating CD8+ T cells are associated with improved prognosis and disease clearance for a variety of cancers and have favorable prognostic value for the patient outcome. For both virus-positive and –negative MCC, the presence of intratumoral CD8+ T cells is associated with improved outcome, reduced likelihood of metastasis and prolonged survival[16, 17].
In the present study, we intended to identify the MHC class I-restricted immunodominant LT epitope and determine if the generation of LT-specific CD8+ T cells could be favored by linking calreticulin (CRT) with LT antigen in the DNA vaccine. CRT exposed on the cell surface of pre-apoptotic cells is a damage-associated molecular pattern (DAMP) recognized by dendritic cells as the green light for phagocytosis and consequently is considered to be a marker of immunogenic cell death. The inclusion of CRT in therapeutic DNA vaccines has been demonstrated to successfully promote the generation of antitumor immune responses in HPV-positive tumor models[20–22], justifying our rationale for the examination of anti-MCC immunotherapy with a DNA vaccine encoding CRT linked to the LT antigen.