Matthew Marr

Date of Award

January 2015

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Peter J. Tattersall

Subject Area(s)

Virology, Oncology, Immunology

Abstract

Melanoma is a prevalent, deadly disease with poor outcomes following metastasis. It is an immunogenic cancer with many unique tumor antigens, but the tumor microenvironment inhibits the immune system. Current monoclonal antibody treatment has been shown to be beneficial in metastatic melanoma by blocking inhibitory immune signals such as PD-1 or CTLA-4. The expression of immunostimulatory proteins such as B7.1 by tumor cells could provide additional immune targeting. Viral vectors based on the parvovirus genome could efficiently and selectively express B7.1 in melanoma cells, such that they become competent to directly activate cognate T cells. Once activated, T cells will become armed as cytotoxic effectors against cells expressing melanoma tumor antigens. To generate a viral vector, we first needed a parvovirus that infected melanoma. Parvoviruses are uniquely adapted to infect tumor cells as viral replication is dependent on the host cell advancing through S phase. However, most parvoviruses tested were unable to establish infection in the murine melanoma cell line B16F10. The most infectious parvovirus in B16F10 was mouse parvovirus 1a (MPV1a). B16F10 cells were infected with MPV1a, and the progeny virus was harvested and used to re-infect more B16F10 cells. After five serial passages of virus, the mutant polyclonal stock named MPV P5 was analyzed. At 24 hours following infection at a multiplicity of 5000 virions per cell, MPV1a infected 16% of cells, whereas MPV P5 infected 74%. Construction of a molecular clone from the polyclonal MPV P5 mixture yielded a construct consisting of the non-structural and virion polypeptide (NS and VP) genes that was able to infect 45% of cells under the same conditions. The mutations that were most responsible for the increase in B16F10 infectivity were isolated to the VP2 region, which encodes the major capsid protein. By incorporating the viral proteins from the clonal version of MPV P5 into a parvoviral vector system, we have generated a vector capable of transducing B16F10 cells and simultaneously expressing the Green Fluorescent Protein marker and B7.1. Further experiments are needed to determine if the B7.1 expression is sufficient at its current rate of infectivity to modulate an immune response, and to examine whether viral products within the tumor cells generate additional immunogenic signaling. Other aspects of the MPV P5 genome may be engineered into the vector to increase its efficiency.

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This is an Open Access Thesis.

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