Induction of P815 tumor immunity by DNA-based recombinant Semliki Forest virus or replicon DNA expressing the P1A gene.

AIM: To compare the prophylactic and therapeutic effects of alphaviruses in the same tumor model, we used a DNA-based approach to generate a replicon DNA and recombinant Semliki Forest virus (rSFV) particles expressing P1A, the P815 mastocytoma tumor associated antigen, and compared the immune effect of each vaccine. METHODS: Six to eight-week-old female DBA/2 mice were inoculated with P1A plasmid or viral vaccines. Spleen cells were assayed for antigen-specific cytotoxic T cell activity. Tumor growth or survival rate was observed in preventive and therapeutic experiments, respectively. RESULTS: We found that the rSFV particles prevented tumor growth when delivered prior to innoculation of mice with P815 cells, and more importantly, improved survival when delivered after the initiation of tumor growth. Naked P1A replicon DNA also functioned as a protective and therapeutic vaccine, although with less potency than rSFV particles. Virus particles also elicited a stronger cellular immune response as measured by target cell lysis. CONCLUSION: rSFV particles have stronger specific prophylactic and therapeutic immune effects in mice than replicon DNA-based DNA vaccines, though the latter is more effective than traditional plasmid vectors (e.g. pCI-neo vector).

Retinoblastoma protein purification and transduction of retina and retinoblastoma cells using improved alphavirus vectors.

PURPOSE: To develop and use improved Semliki Forest vectors (SFVs) for functional and structural analyses of the retinoblastoma protein (RB) in developing retina and retinoblastoma cells. METHODS: Virus was harvested from cells transfected with replicon and helper plasmids. Combinations of producer and target cells were tested for optimal virus production and protein expression. The replicon was improved by adding an expanded multiple cloning site, translational enhancer, and FLAG and HIS10 epitope and affinity tags. Affinity chromatography was used to purify beta-galactosidase or RB. RB function was assessed through interaction with E2F1. The efficacy of SFV as a retinal delivery system was tested on mouse explants and cultured human retinoblastoma cells. RESULTS: The optimal producer and target cell lines were an HEK-293 derivative (Phoenix Eco) and BHK-21, respectively. Stable expression of structural proteins in the BHK-21 helper line simplified virus production and amplified virus yield 100-fold. The translational enhancer improved expression three- to eightfold. Full-length, functional RB was produced without the truncated products characteristic of bacterially produced RB and was purified using the affinity tags. SFVs efficiently transduced mouse retinal explants and multiple hard-to-transduce retinoblastoma tumor lines. CONCLUSIONS: This study describes a simple, rapid, SFV vector system to produce recombinant proteins, such as RB, in mammalian cells. These SFV vectors represent an efficient approach to purification of proteins and protein complexes and transduction of retinal or retinoblastoma cells for the purpose of in vivo analysis of protein functions.