Studies on persistent polyomavirus infection in relation to tumor development and options for vaccine and gene therapy

Abstract

Polyomavirus is a small DNA virus capable of inducing tumors in mice with a defective or an immature immune system. The importance of T-cells in prevention of polyoma-induced tumor development has previously been shown. In this thesis, we wished to elucidate the role of humoral immunity with regard to polyomavirus persistence and tumor development. Adult and newborn X-linked immunodeficient (XID) mice with a reduced antibody response and µMT (also referred to as IgM-/-) mice, which lack functional antibodies, were infected with polyomavirus. The mice were examined for persistence of viral DNA by a polyoma specific PCR and for presence of tumors. A disseminated persistent polyomavirus infection was observed in all µMT mice with a severe B-cell deficiency, while in XID mice with a less severe defect, the infection was more limited but still persistent. There was no major difference in tumor frequency between B-cell deficient and normal mice, confirming the importance of T-cells in prevention of tumors.

For future gene therapy applications, a safe and efficient vector able to introduce DNA into cells in vivo is desirable. The murine polyomavirus major capsid protein VP1 can self assemble, enclose eukaryotic DNA and transfer the DNA into cells in vitro. We aimed to examine the efficiency of VP1-pseudocapsids to transfer exogenous DNA into cells in vivo. Polyoma VP1-pseudocapsids, obtained from a baculovirus system, were used to introduce a replication defective polyoma DNA into cells in vivo. This process was carried out in normal, B-cell deficient (µMT) and T-cell deficient (CD4-/-8-/-) mice on C57BL/6 background. DNA transfer by VP1-pseudocapsids was compared to introduction of naked DNA or to polyomavirus infection. Polyoma DNA was successfully introduced by VP1-psedudocapsids into a variety of tissues, where it remained for at least 3-6 months, which was comparable to that observed after polyomavirus infection. Quantification of the introduced DNA by real time PCR revealed that introduction of DNA by VP1-pseudocapsids in normal mice was comparable to that by natural virions. In search for a more restricted gene transfer vector a novel murine polyomavirus, BG, previously reported to target the nervous system, was compared to the polyomavirus A2 strain. Tissue distribution and persistence of BG was not restricted to the nervous system as anticipated, but was spread widely similar to A2. In addition, BG demonstrated a more potent oncogenic profile by inducing multiple tumors shortly after infection in newborn CBA mice, whereas A2 infected mice developed single tumors and during a longer latency period.

Finally, we designed a study to examine the possibility to immunize T-cell deficient CD4-/-8-/- mice against polyomavirus infection by using viral pseudocapsids and temperature sensitive mutants as immunogens. Pre-immunization of polyomavirus infected mice resulted in a partial protective response against polyomavirus infection in contrast to a persistent viral infection, which was obtained in polyomavirus infected non-immunized controls.