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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

Characterization of ST-specific CD8+ T cells using splenocytes stimulated with ST peptide aa 19-27. (A) Outline of the vaccination schedule. C57BL/6 mice (5 per group) were immunized with pcDNA3-MCC/ST intradermally with DNA-coated particles using a helium-driven gene gun 3 times at 4-day intervals. Pooled splenocytes from vaccinated mice were collected and cultured in vitro with overlapping ST peptide overnight, and stained for intracellular IFN-γ and CD8+ cell surface marker. (B) Intracellular cytokine staining followed by flow cytometry analysis to characterize ST-specific CD8+ T cell epitope using a single ST peptide (aa 19-27) from splenocytes harvested from mice vaccinated with pcDNA3-MCC/ST or pcDNA3. (C) Bar graph of representative flow cytometry data showing the number of ST-specific CD8+ T cells among 3 × 105 splenocytes. Note that peptide 19-27 activated the highest number of ST-specific CD8+ T cells.
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Figure 2: Characterization of ST-specific CD8+ T cells using splenocytes stimulated with ST peptide aa 19-27. (A) Outline of the vaccination schedule. C57BL/6 mice (5 per group) were immunized with pcDNA3-MCC/ST intradermally with DNA-coated particles using a helium-driven gene gun 3 times at 4-day intervals. Pooled splenocytes from vaccinated mice were collected and cultured in vitro with overlapping ST peptide overnight, and stained for intracellular IFN-γ and CD8+ cell surface marker. (B) Intracellular cytokine staining followed by flow cytometry analysis to characterize ST-specific CD8+ T cell epitope using a single ST peptide (aa 19-27) from splenocytes harvested from mice vaccinated with pcDNA3-MCC/ST or pcDNA3. (C) Bar graph of representative flow cytometry data showing the number of ST-specific CD8+ T cells among 3 × 105 splenocytes. Note that peptide 19-27 activated the highest number of ST-specific CD8+ T cells.

Mentions: For the characterization of the ST-specific CD8+ T cell immune response, DNA-coated particles were delivered to the shaved abdominal region of each mouse by a helium-driven gene gun. The vaccination schedule is outlined in Figure 2A. The DNA vaccine we generated encoded MCPyV ST aa 1-186 (pcDNA3-MCC/ST). Empty pcDNA3 vector was used as a control. To determine whether vaccination of pcDNA3-MCC/ST or pcDNA3 vaccine was capable of generating ST-specific CD8+ T cell immune responses, intracellular cytokine staining for IFN-γ was performed, followed by flow cytometry. As shown in Figure 2B and C, mice vaccinated with pcDNA3-MCC/ST generated a significant ST-specific CD8+ T cell response when stimulated with the peptide spanning aa 19-27. In comparison, mice vaccinated with the empty vector pcDNA3 did not generate significant numbers of ST-specific CD8+ T cell-mediated responses when stimulated with the same peptides. Thus, our data show that mice vaccinated with MCPyV ST DNA could be generate potent ST-specific CD8+ T cell-mediated immune responses.


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)

Characterization of ST-specific CD8+ T cells using splenocytes stimulated with ST peptide aa 19-27. (A) Outline of the vaccination schedule. C57BL/6 mice (5 per group) were immunized with pcDNA3-MCC/ST intradermally with DNA-coated particles using a helium-driven gene gun 3 times at 4-day intervals. Pooled splenocytes from vaccinated mice were collected and cultured in vitro with overlapping ST peptide overnight, and stained for intracellular IFN-γ and CD8+ cell surface marker. (B) Intracellular cytokine staining followed by flow cytometry analysis to characterize ST-specific CD8+ T cell epitope using a single ST peptide (aa 19-27) from splenocytes harvested from mice vaccinated with pcDNA3-MCC/ST or pcDNA3. (C) Bar graph of representative flow cytometry data showing the number of ST-specific CD8+ T cells among 3 × 105 splenocytes. Note that peptide 19-27 activated the highest number of ST-specific CD8+ T cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Characterization of ST-specific CD8+ T cells using splenocytes stimulated with ST peptide aa 19-27. (A) Outline of the vaccination schedule. C57BL/6 mice (5 per group) were immunized with pcDNA3-MCC/ST intradermally with DNA-coated particles using a helium-driven gene gun 3 times at 4-day intervals. Pooled splenocytes from vaccinated mice were collected and cultured in vitro with overlapping ST peptide overnight, and stained for intracellular IFN-γ and CD8+ cell surface marker. (B) Intracellular cytokine staining followed by flow cytometry analysis to characterize ST-specific CD8+ T cell epitope using a single ST peptide (aa 19-27) from splenocytes harvested from mice vaccinated with pcDNA3-MCC/ST or pcDNA3. (C) Bar graph of representative flow cytometry data showing the number of ST-specific CD8+ T cells among 3 × 105 splenocytes. Note that peptide 19-27 activated the highest number of ST-specific CD8+ T cells.
Mentions: For the characterization of the ST-specific CD8+ T cell immune response, DNA-coated particles were delivered to the shaved abdominal region of each mouse by a helium-driven gene gun. The vaccination schedule is outlined in Figure 2A. The DNA vaccine we generated encoded MCPyV ST aa 1-186 (pcDNA3-MCC/ST). Empty pcDNA3 vector was used as a control. To determine whether vaccination of pcDNA3-MCC/ST or pcDNA3 vaccine was capable of generating ST-specific CD8+ T cell immune responses, intracellular cytokine staining for IFN-γ was performed, followed by flow cytometry. As shown in Figure 2B and C, mice vaccinated with pcDNA3-MCC/ST generated a significant ST-specific CD8+ T cell response when stimulated with the peptide spanning aa 19-27. In comparison, mice vaccinated with the empty vector pcDNA3 did not generate significant numbers of ST-specific CD8+ T cell-mediated responses when stimulated with the same peptides. Thus, our data show that mice vaccinated with MCPyV ST DNA could be generate potent ST-specific CD8+ T cell-mediated immune responses.

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