406. Role of MyD88 Signaling Pathway in the Host Immune Responses in Adenoviral Vector-Mediated Gene Therapy

Abstract

We investigated the role of MyD88-mediated TLR signaling pathways in the acute and adaptive immune responses to systematic administration of human Ad5 in the gene therapy setting. Following intravenous administration of E1E3-deleted version of Ad5-lacZ (2E11 pt. /animal) to both wild type C57B6 and MyD88 knockout (KO) mice, serum inflammatory cytokines including IL6, KC and TNF- were detected robustly in C57B6 mice but remained barely detectable in MyD88 KO mice. Accordingly immunostaining using CD11b antibody on liver sections at 6h showed a robust infiltration of inflammatory neutrophil cells into the liver, while few was observed in the livers of the MyD88 KO mice. A previous study in our lab has shown that dendritic cells are among the sources of the acute cytokine response (Zhang Y et. al. Molecular Therapy 2001, Vol. 3, p697). To further understand the molecular role of MyD88, CD11c+ dendritic cells were enriched from the spleens using a magnetic cell-sorting system (Miltenyi Biotech, CA). At 6 h after the vector infusion, a strong spontaneous secretion of all the three inflammatory cytokines was seen in the in vitro culture of the CD11c+ DCs isolated from C57B6 mice, but none was detectable in cells from MyD88 KO mice. Furthermore, adaptive immune responses such as antibody and T cell responses were studies. At day 14 after Ad5-LacZ infusion, total immunoglobulins in the serum against the vector and transgene lacZ were all significantly attenuated in the KO mice (ELISA); the virus blocking titer of MyD88 serum was almost diminished compared to that of C57B6 (Neutralizing antibody assay). Mice were intramuscularly administrated with Ad5-Spike, the frequency of INF- producing CD8+ T cells in response to a Spike CD8+ epitope were reduced by 50% in MyD88 KO mice compared to C57B6 mice (INF- intracellular staining assay); T cell proliferation in response to Ad5 particles (H3 Thymidine incorporation assay) and cytokine production by whole spleen cells (ELISA) was also attenuated in the KO mice. Finally, LacZ staining on liver sections showed a prolonged transgene expression in MyD88 (100% at D28; 90% at D50) compared to C57B6 (1% at D28; 0% at D50) mice. In summary, the host immune responses were greatly blunted and the duration of transgene expression was significantly extended when MyD88-mediated TLR signaling is abolished, which suggests a promising alternative towards improving Ad-mediated gene therapy.