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

Gomez B, He L, Tsai YC, Wu TC, Viscidi RP, Hung CF - Cell Biosci (2013)

Bottom Line: 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.The LT-targeting DNA vaccine generated prolonged survival, decreased tumor size and increased LT-specific CD8+ T cells in tumor-bearing mice.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA. chung2@jhmi.edu.

ABSTRACT

Background: Merkel 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.

Results: In 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.

Conclusions: The 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.

No MeSH data available.


Related in: MedlinePlus

Generation and characterization of ST-expressing B16/ST tumor cell line. B16 mouse melanoma cells were transduced with a lentiviral vector containing a mammalian codon-optimized gene encoding Merkel cell polyomavirus (strain 350) small T antigen (ST) under the control of cytomegalovirus and GFP reporter under EF1 promoter to generate tumorigenic B16/ST tumor cell line. (A) Characterization of the transduction of B16/ST tumor cells by flow cytometry analysis after sorting. B16/ST tumor cells (green) or control B16 melanoma cells (purple) were sorted and characterized for GFP expression by flow cytometry analysis. (B) Schematic diagram of vaccination schedule for Western Blot analysis. C57BL/6 mice were vaccinated intramuscularly by electroporation three times at 1-week intervals and boosted at the same dose. Western Blot analysis using sera from vaccinated mice was performed 1 month after last vaccination to determine ST protein levels of B16/ST cells. (C) ST protein levels determined by Western blot analysis. Membranes were probed with either serum from mice vaccinated with pcDNA3-MCC/ST or anti-β-actin antibody for loading control. Lane 1, negative control B16. Lane 2, B16-MCC/ST. (D) ST RNA levels determined by RT-PCR. Lane 1, negative control B16. Lane 2, B16-MCC/ST.
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Figure 1: Generation and characterization of ST-expressing B16/ST tumor cell line. B16 mouse melanoma cells were transduced with a lentiviral vector containing a mammalian codon-optimized gene encoding Merkel cell polyomavirus (strain 350) small T antigen (ST) under the control of cytomegalovirus and GFP reporter under EF1 promoter to generate tumorigenic B16/ST tumor cell line. (A) Characterization of the transduction of B16/ST tumor cells by flow cytometry analysis after sorting. B16/ST tumor cells (green) or control B16 melanoma cells (purple) were sorted and characterized for GFP expression by flow cytometry analysis. (B) Schematic diagram of vaccination schedule for Western Blot analysis. C57BL/6 mice were vaccinated intramuscularly by electroporation three times at 1-week intervals and boosted at the same dose. Western Blot analysis using sera from vaccinated mice was performed 1 month after last vaccination to determine ST protein levels of B16/ST cells. (C) ST protein levels determined by Western blot analysis. Membranes were probed with either serum from mice vaccinated with pcDNA3-MCC/ST or anti-β-actin antibody for loading control. Lane 1, negative control B16. Lane 2, B16-MCC/ST. (D) ST RNA levels determined by RT-PCR. Lane 1, negative control B16. Lane 2, B16-MCC/ST.

Mentions: B16 mouse melanoma cells were transduced with a lentiviral vector containing a mammalian gene encoding MCPyV ST antigen under the control of cytomegalovirus (CMV) promoter and GFP reporter under EF1 promoter to generate a tumorigenic ST-expressing cell line, B16/ST. Successful transduction of the lentivirus was confirmed by high levels of GFP expression, which allowed us to isolate the transduced tumor cells. As shown in Figure 1A, the ST lentivirus-transduced B16 tumor cells demonstrated significantly higher GFP expression levels compared to non-transduced B16 tumor cells. The GFP-positive cells were further isolated for the characterization of the expression of ST antigen. In order to generate antibodies against ST antigen, mice were vaccinated with pcDNA3-MCC/ST intramuscularly by electroporation according to the schedule shown in Figure 1B. One month following the last vaccination, sera from vaccinated mice were collected for Western blot and RNA analysis. As shown in Figure 1C and D, cell lysates from B16/ST cells demonstrated a specific band consistent with ST antigen protein and RNA levels. In comparison, cell lysates from B16 melanoma cells did not show such a band, indicating the absence of ST antigen. To ensure equal loading of cell lysates, β-actin was used as a control. Thus, our data show the successful creation of a murine B16 tumor cell line that expresses ST, B16/ST.


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

Gomez B, He L, Tsai YC, Wu TC, Viscidi RP, Hung CF - Cell Biosci (2013)

Generation and characterization of ST-expressing B16/ST tumor cell line. B16 mouse melanoma cells were transduced with a lentiviral vector containing a mammalian codon-optimized gene encoding Merkel cell polyomavirus (strain 350) small T antigen (ST) under the control of cytomegalovirus and GFP reporter under EF1 promoter to generate tumorigenic B16/ST tumor cell line. (A) Characterization of the transduction of B16/ST tumor cells by flow cytometry analysis after sorting. B16/ST tumor cells (green) or control B16 melanoma cells (purple) were sorted and characterized for GFP expression by flow cytometry analysis. (B) Schematic diagram of vaccination schedule for Western Blot analysis. C57BL/6 mice were vaccinated intramuscularly by electroporation three times at 1-week intervals and boosted at the same dose. Western Blot analysis using sera from vaccinated mice was performed 1 month after last vaccination to determine ST protein levels of B16/ST cells. (C) ST protein levels determined by Western blot analysis. Membranes were probed with either serum from mice vaccinated with pcDNA3-MCC/ST or anti-β-actin antibody for loading control. Lane 1, negative control B16. Lane 2, B16-MCC/ST. (D) ST RNA levels determined by RT-PCR. Lane 1, negative control B16. Lane 2, B16-MCC/ST.
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Figure 1: Generation and characterization of ST-expressing B16/ST tumor cell line. B16 mouse melanoma cells were transduced with a lentiviral vector containing a mammalian codon-optimized gene encoding Merkel cell polyomavirus (strain 350) small T antigen (ST) under the control of cytomegalovirus and GFP reporter under EF1 promoter to generate tumorigenic B16/ST tumor cell line. (A) Characterization of the transduction of B16/ST tumor cells by flow cytometry analysis after sorting. B16/ST tumor cells (green) or control B16 melanoma cells (purple) were sorted and characterized for GFP expression by flow cytometry analysis. (B) Schematic diagram of vaccination schedule for Western Blot analysis. C57BL/6 mice were vaccinated intramuscularly by electroporation three times at 1-week intervals and boosted at the same dose. Western Blot analysis using sera from vaccinated mice was performed 1 month after last vaccination to determine ST protein levels of B16/ST cells. (C) ST protein levels determined by Western blot analysis. Membranes were probed with either serum from mice vaccinated with pcDNA3-MCC/ST or anti-β-actin antibody for loading control. Lane 1, negative control B16. Lane 2, B16-MCC/ST. (D) ST RNA levels determined by RT-PCR. Lane 1, negative control B16. Lane 2, B16-MCC/ST.
Mentions: B16 mouse melanoma cells were transduced with a lentiviral vector containing a mammalian gene encoding MCPyV ST antigen under the control of cytomegalovirus (CMV) promoter and GFP reporter under EF1 promoter to generate a tumorigenic ST-expressing cell line, B16/ST. Successful transduction of the lentivirus was confirmed by high levels of GFP expression, which allowed us to isolate the transduced tumor cells. As shown in Figure 1A, the ST lentivirus-transduced B16 tumor cells demonstrated significantly higher GFP expression levels compared to non-transduced B16 tumor cells. The GFP-positive cells were further isolated for the characterization of the expression of ST antigen. In order to generate antibodies against ST antigen, mice were vaccinated with pcDNA3-MCC/ST intramuscularly by electroporation according to the schedule shown in Figure 1B. One month following the last vaccination, sera from vaccinated mice were collected for Western blot and RNA analysis. As shown in Figure 1C and D, cell lysates from B16/ST cells demonstrated a specific band consistent with ST antigen protein and RNA levels. In comparison, cell lysates from B16 melanoma cells did not show such a band, indicating the absence of ST antigen. To ensure equal loading of cell lysates, β-actin was used as a control. Thus, our data show the successful creation of a murine B16 tumor cell line that expresses ST, B16/ST.

Bottom Line: 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.The LT-targeting DNA vaccine generated prolonged survival, decreased tumor size and increased LT-specific CD8+ T cells in tumor-bearing mice.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA. chung2@jhmi.edu.

ABSTRACT

Background: Merkel 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.

Results: In 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.

Conclusions: The 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.

No MeSH data available.


Related in: MedlinePlus