The premise of this project was to engineer a novel viral platform with the capacity to enhance antitumour immunity. To this effect, the N1L gene was disrupted in a Lister strain vaccinia viral backbone that had previously been engineered to be tumour selective (VVL15ΔN1L) and armed with transgenes encoding murine and human versions of GMCSF and IL12. In vitro, they retained potency for infecting, replicating in and killing a panel of murine, Syrian hamster and human cancer cells; all viruses were able to express their transgenes to detectable levels upon infection of every tumour cell line. In comparison to the parental virus (VVL15), VVL15ΔN1L administration into immune competent in vivo tumour models (of pancreatic and lung cancer) led to enhanced intratumour (IT) infiltration of neutrophils as well as markedly elevated circulating numbers of natural killer (NK) cells. VVL15ΔN1L also enhanced the tumour infiltration of CD8+ cells. Functional immunoassays and flow cytometric analysis of T cells provided evidence of enhanced tumour specific adaptive immunity. In comparison to VVL15, IT VVL15ΔN1L significantly reduced the growth of subcutaneously implanted syngeneic pancreatic tumours. This effect was predominantly due to cytotoxic lymphocytes, evidenced by the complete abrogation of efficacy upon repeating the experiment in mice that had been depleted of CD8+ cells. A similar treatment schedule reduced the formation of lung metastases from a primary spontaneously metastasising syngeneic lung cancer model; and translated into prolonged short-term post-operative survival when used as neoadjuvant to surgical resection. Efficacy in this context was contrastingly, due to an elevation in systemic NK cells; concurrent depletion of NK cells (but not CD4+ or CD8+ cells) completely abrogated the survival advantage. The IL12 transgene armed recombinant was the most effective antitumour therapeutic. Its IT administration into pancreatic tumours led to complete tumour eradication in over 80% of tumour bearing mice and was effective in slowing the growth of other aggressive flank tumours. Neoadjuvant administration of VVL15ΔN1L-mIL12 into metastatic lung cancers dramatically prolonged long-term post-surgical survival, with apparent cure of 88% of mice.

Chapter 1: Role of JEV NS1 in protective immunity and in immunopathology. Previous studies from our laboratory revealed T cell immunodominance of non structural proteins NS3 and NS1 during natural JEV infections in humans where as the structural protein E, which is a good target for neutralizing antibody response is a poor inducer of T cells. Flavivirus NS1 is also known to induce humoral immune response. Several studies in different flaviviruses have indicated a role for NS1-specific immune responses in protection against flaviviruses. Paradoxically, studies also pointed to the contribution of NS1 in pathology and immune modulation. We screened serum samples from 72 convalescent JE patients for the presence of anti-NS1 antibodies by ELISA and radioimmunoprecipitation and found NS1 reactivity in 45 samples. These antibodies to NS1 are capable of inducing complement mediated cytolysis of cells expressing NS1 on the surface. Additionally, we demonstrated twenty two fold reduction in the infectious virus produced at 48h in SW-13 cells in the presence of human complement and NS1 antiserum compared to control serum, suggesting that complement mediated cytolytic activity of anti NS1 antibody helps the host in controlling the virus propagation. Chapter 2: Comparison of immune responses to JEV structural proteins Capsid and Envelope in human volunteers vaccinated with inactivated JE vaccine and naturally exposed to live JEV. We compared the CMI responses to structural proteins E and C in human volunteers vaccinated with commercially available killed JE vaccine and in humans naturally exposed to live JEV. The results revealed that structural proteins E and C are inherently poor inducers of T cells even in killed vaccine preparation, where there is no competition from immunodominant non structural proteins. Therefore inclusion of nonstructural proteins NS1 and NS3 along with neutralizing antibody inducing envelope should improve memory and efficacy of a JE vaccine. Chapter 3: Construction and testing in the mouse model of experimental recombinant poxvirus vaccines expressing prM, E, NS1, and NS3 of JEV. Guided by the information on immune responses to JEV in the JE endemic human cohort and volunteers vaccinated with killed JE vaccine, we designed experimental vaccines as recombinant vaccinia viruses expressing NS1, NS3, prM, and E proteins of JEV (vNS1NS3prME) or NS1, NS3, prM, and C-terminally truncated E (vNS1NS3prMΔE) and studied the immune responses elicited by these vaccines in mice. Our data showed that a recombinant vaccinia virus expressing prM, ΔE, NS1, and NS3 of JEV is superior to killed JE vaccine in eliciting long lived neutralizing antibodies as well as NS1 and NS3-specific cytotoxic T lymphocytes (CTL) in addition to NS1-specific cytolytic antibodies, resulting in long lasting and enhanced protection from lethal JEV infection in mice. Our results thus identified all B and T cell antigens whose inclusion in a live-vectored vaccine would provide a vaccine with far superior efficacy over the inactivated JE vaccine.