457. Both Regulatory T and B Cells May Play Roles in Systemic Immune Tolerance Induced by rAAV-Mediated Gene Transfer to Liver

Abstract

The intramuscular delivery of vectored antibody by recombinant adeno-associated virus (rAAV) holds promise for preventing and treating emerging infectious diseases. Recent studies in nonhuman primates suggested that a single intramuscular injection of rAAV1 expressing anti-SIV neutralizing antibody provided long-term protection from SIV infection. However, some of NHP recipients generated anti-antibody responses that eliminate neutralizing activity of vectored antibodies. We exploit different strategies to engineer AAV vectors that can surmount such anti-antibody responses, one of which is to induce systemic tolerance to vectored antibody. It is well established that the hepatic gene transfer can induce systemic immune tolerance to transgene products. Here, we attempted to use hepatotropic AAV serotypes and hepatocyte-specific promoter for liver-specific gene expression to induce systemic immune tolerance to muscle-expressed transgene products including intracellular β-Galactosidase (β-gal) or secreted Ovalbumin (OVA). First, we tested if the transgene specific CD8+ T cells could be reduced by using liver-specific promoter. C57BL/6 mice were intravenously delivered with rAAV9 vectors expressing β-gal from either ubiquitous chicken β-actin (CB) promoter or liver-specific human thyroxine binding globulin (TBG) promoter. When compared to CB promoter, TBG can achieve significantly higher β-gal expression with reduced β-gal specific CD8+ T cells. To compare the humoral immune responses against the secrete transgene products, AAV8 vectors expressing OVA from CB or TBG were intravenously injected to C57BL/6 mice. AAV8.TBG.OVA injected mice showed significant higher OVA expression than AAV8.CB.OVA injected mice did. Interestingly, while the mice received rAAV1CB.OVA intramuscularly triggered robust anti-OVA IgG1 response with low and transient OVA expression, such responses in the mice treated with intravenously delivered both AAV8 vectors were negligible accompanied with high levels of sustained OVA expression. In addition, the high frequency of regulatory T cells and CD19+CD5+CD1d+ regulatory B cells are detected in the spleens from the mice intravenously delivered both AAV8OVA vectors. Finally, we tested whether hepatic restricted OVA gene transfer by AAV8.TBG.OVA can indeed induce immune tolerance to subsequently and intramuscularly delivered AAV1.CB.OVA in C57BL/6 mice. Again, very low levels of transient OVA expression and high levels of anti-OVA IgG1 were detected in the animals treated with PBS followed by intramuscular AAV1.CB.OVA, whereas the intravenously delivered AAV8.TBG.OVA vectors facilitated the subsequently muscle-directed AAV1-encoded OVA expression without antibody production. Our data suggest that both regulatory T and B cells may play roles in systemic immune tolerance induced by liver-derived transgene expression from liver tropic AAV vector.