In recent years, Non-Hodgkin's lymphoma (NHL) has risen to be the most common hematologic malignancy in the United States. Aggressive new combination chemotherapy regimes have slowly improved remission rates for most stages of disease, however the risk of relapse is still significant, even if patients achieve remission during therapy, and patients that relapse are characterized by a reduced response to treatment. To improve patient survival outcomes, significant attention has been focused on the generation of an active, tumor specific, immune response through vaccination with a tumor-specific antigen: the cell-surface expressed immunoglobulin (Ig), or idiotype. Clinical studies show that when patients make an immune response to the vaccine, they have improved survival characteristics. Protein Ig, however, is difficult to make for patient specific applications, and may not provide adequate immune stimulation in tumor-bearing patients. Our hypothesis is that RNA based vaccines can improve anti-idiotype immunity by delivering idiotype sequences in the context of viral antigen delivery, in a system that is ideally suited for simple and rapid individualized vaccine production. Our goal is to design and test model antigen expressing RNA constructs to determine the efficacy of RNA based idiotype vaccines in two murine models of NHL (38C13 and A20). These models are ideal, because they require either antibody (38C13) or CTL activation (A20) for optimal tumor protection, characteristics that are also needed for successful patient immune activation. We will use Semliki Forest Virus (SFV) nonstructural genes to drive expression of 38C13 and A20 idiotype sequences, either singly or in tandem with cytokine gene sequences for maximum antigen delivery and immune activation. SFV RNA, rather than encapsidated in cell culture with native capsid, will be encapsidated using the novel self-assembly properties of Tobacco Mosaic Virus coat protein. Preparation of encapsidated RNA vaccines is simple, involving merely mixing coat protein with RNA to generate fully protected pseudovirus particles. Encapsidated RNA pseudovirus vaccines induce both antibody and cytotoxic T lymphocyte (CTL) activation, as well as tumor protective immunity, without additional adjuvants. Vaccine agents can be "cloaked" with surface peptides reduce immune responses to viral coat protein, and are stable for repeated vaccine antigen delivery and boosting. We will establish the best method to co-deliver cytokine genes to promote improved immune activation and reversal of self-antigen tolerance to 38C13 and A20 NHL tumor antigens. Antigen expression level and cytokine activity will be verified in vitro, as well as antibody and CTL reactivity to target antigens in vivo. We will test peptide antigen conjugates that may boost anti-idiotype immunity or improve vaccine potency. Lastly, we will test the most effective idiotype antigen/cytokine encapsidated RNA formulations in vivo and correlate improved immunity with tumor protection in the relevant 38C13 or A20 mouse tumor model. PUBLIC HEALTH RELEVANCE: Improving cancer vaccine therapy by rational drug design is a high priority of research scientists and the medical community. This R21 application seeks to implement improved cancer vaccine characteristics by harnessing innate immunity to RNA viruses. Our hypothesis is that encapsidated RNA vaccines that express non-Hodgkin's lymphoma tumor antigens will provide superior immune activation and protection against lymphoma tumor growth. Unlike current therapies, custom vaccines are easily made, and may facilitate widespread adoption of individualized patient immunotherapy.