Adeno-Associated Virus Capsid Structure Drives CD4-Dependent CD8+ T Cell Response to Vector Encoded Proteins

Abstract

The immunological sequelae of adeno-associated virus (AAV)-mediated gene transfer in vivo is quite complex. In murine models, most AAV capsids are associated with minimal or dysfunctional T cell responses to antigenic transgene products. In this study we compared T cell activation against AAV2/8 and AAV2/rh32.33 vectors expressing nuclear-targeted LacZ (nLacZ), GFP, or firefly luciferase in murine skeletal muscle. We show that, unlike AAV8, AAVrh32.33 yields qualitatively and quantitatively robust T cell responses to both the capsid and transgene product. AAV2/rh32.33.CB.nLacZ, but not AAV2/8, drives a high degree of cellular infiltration and a loss of detectable transgene expression in C57BL/6 mice. However, cellular immunity to AAVrh32.33 is ablated in the absence of CD4, CD40L, or CD28, permitting stable β-galactosidase expression. Treatment of CD40L−/− mice with the CD40 agonist, FGK45, failed to restore the CD8 response to AAV2/rh32.33.nLacZ, suggesting that additional factors are involved. Our results suggest that specific domains within the AAVrh32.33 capsid augment the adaptive response to both capsid and transgene Ags in a CD4-dependent pathway involving CD40L signaling and CD28 costimulation. Structural comparison of the AAV8 and rh32.33 capsids has identified key differences that may drive differential immunity by affecting tropism, Ag presentation or the activation of innate immunity. This murine model of AAV-mediated cytotoxicity allows us to delineate the mechanism of viral immune activation, which is relevant to the translation of AAV technology in higher order species.

CpG-depleted adeno-associated virus vectors evade immune detection

“…Studies in C57BL/6 mice demonstrate that intramuscular gene transfer with AAVrh32.33 vectors induces a robust adaptive immune response toward both capsid and transgene antigen, heavy cellular infiltrate, and a loss of detectable transgene expression at days 35 and 60 after administration (18). To evaluate the role of TLR9 signaling in the induction of this adaptive immune response and transgene loss, WT and Tlr9-deficient mice were i.m.…”

“…Heavy CD4 + and CD8 + cellular infiltrate has been observed in WT C57BL/6 mice following AAVrh32.33 intramuscular vector transduction (18). To determine the requirement for TLR9 signaling in the induction of this extensive infiltrate, WT and Tlr9-KO muscle cryosections were stained with anti-CD4 and anti-CD8 Abs and examined by fluorescent microscopy at 35 and 60 days after vector administration ( Figure 3A).…”

“…Historically, AAV8 gene delivery to WT C57BL/6 muscle tissue results in minimal Th1 responses, negligible cellular infiltrate and prolonged transgene expression (18). To investigate the role of TLR9 detection of AAV8 and its effect, if any, on AAV8 gene expression, WT and Tlr9-KO mice were injected i.m.…”

“…We compared responses from WT recipients with responses from TLR9 mice that received vectors based on the immunogenic capsid from AAVrh32.33 (18). AAVrh32.33 vectors stimulate a robust transgene and capsid T cell response that results in detectable loss of transgene expression.…”

Duchenne muscular dystrophy gene therapy in the canine model

“…124 Recently, several laboratories compared the immunity of AAV-8 to that of other AAV serotypes (AAV-1, 2, and 5 and rh32.33). 124,131,[135][136][137][138][139][140] Interestingly, all these studies suggest that AAV-8 is less immunogenic. Future mechanistic studies may reveal the molecular underpinning of the unique immune privilege demonstrated by AAV-8.…”