IMMUNE-MEDIATED DESTRUCTION OF TRANSFECTED MUSCLE-FIBERS AFTER DIRECTGENE-TRANSFER WITH ANTIGEN-EXPRESSING PLASMID DNA

DNA-based immunization of mice by intramuscular injection antigen-enco ding plasmid DNA results in immune responses which may be sustained fo r extended periods of time without an antigen boost. For example, we h ave previously shown that a strong humoral response against hepatitis B virus surface antigen (HBsAg) will persist for up to 74 weeks follow ing a single intramuscular administration of DNA. It has been proposed that the longevity of the response is due to sustained expression of antigen in transfected muscle cells. However, here we show by immunohi stochemistry an electron microscopy that HBsAg-expressing muscle fiber s are destroyed around 10 days after injection of DNA in mice. We have also evaluated destruction of the transfected muscle fibers indirectl y, by measurement of luciferase activity in muscles at differ ent time s after injection of a luciferase reporter gene construct, alone or in combination with HBsAg-expressing DNA. Control muscles injected with luciferase-expressing DMA alone maintain expression of high levels of luciferase for at least 60 days. In contrast, muscles co-injected with DNAs expressing luciferase and a secreted form of HBsAg show high lev els of luciferase activity at 5 days but >99% of this is lost by 20 da ys. Similar results are obtained with co-expression of luciferase and beta-galactosidase, a nonsecreted antigen. toss of luciferase expressi on does not occur in muscles of mice with severe combined immunodefici ency, indicating that the myofiber destruction is immunologically medi ated.