Induction of CTL responses by plasmid DNA immunization

by Ming, Marin

Abstract (Summary)
Vaccination is the most effective public health measure yet devised to decrease human morbidity and mortaiity. Despite succeu at controiiing some pathogens. current vaccination techniques have proven ineffective for the treatment of many important diseases. A novel method of vaccination has recently been discovered. and is based on the ability of injected plasrnid DNA to be taken up and expressed by celis in situ in such a way as to result in systemic immunity to the encoded gene product. The mechanisms by which DNA vaccines initiate cellular immunity are unclear. Bone marrow-denved antigen pnsenting cells are known to be responsible for the initiation of cellular immunity. but the question of how these cells acquire antigen remains to be solved. APCs are either directly transfated during the vaccination process, or they acquire antigen from an exogenous source and process it for direct presentation to T cells. Experiments described here address this question of induction of cellular immunity by DNA vaccines. The form of possible protein transfer was investigaied: mice were immunized with influenza NP protein to elicit anti-NP antibodies, then immunized with plasmid DNA encoding the NP protein; the DNA vaccine-induced CTL response was found to be uninhibited by the presence of preexisting ami NP antibodies. These results suggest that protein is not transferred in a 'naked' form from msfected ceiis to APCs. A novel method for selecting in vitro transfected ceiƮs is described, based upon FACS sorting of fiuorescently labeled gold particles used for transfection. To investigaie the role of transfected APCs, ceils with dendritic phenotype were cuitured from murine bone marrow, ttansfected in vitro, and transferred to naive recipients. Attempts to induce immunity to the transfected gene product using this approach were unsuccessful. An experimental design is outlined for the detennination of the balance between protein transfer and direct transfection of APCs in the case of gene gun immunization, utihg bone marrow dekd dendritic ceils and in vitro gene gun transfection.
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Source Type:Master's Thesis



Date of Publication:01/01/1999

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