Unique Roles of TLR9- and MyD88-Dependent and -Independent Pathways in Adaptive Immune Responses to AAV-Mediated Gene Transfer

Abstract

The immune system represents a significant barrier to successful gene therapy with adeno-associated viral (AAV) vectors. In particular, adaptive immune responses to the viral capsid or the transgene product are of concern. The sensing of AAV by toll-like receptors (TLRs) TLR2 and TLR9 has been suggested to play a role in innate immunity to the virus and may also shape subsequent adaptive immune responses. Here, we investigated the functions of TLR2, TLR9 and the downstream signaling adaptor MyD88 in antibody and CD8+ T-cell responses. Antibody formation against the transgene product occurred largely independently of TLR signaling following gene transfer with AAV1 or AAV2 vectors, whereas loss of signaling through the TLR9-MyD88 pathway substantially reduced CD8+ T-cell responses. In contrast, MyD88 (but neither of the TLRs) regulated antibody responses to capsid. B cell-intrinsic MyD88 was required for the formation of anti-capsid IgG2c independently of vector serotype or route of administration. However, MyD88^-/- mice instead produced anti-capsid IgG1 that emerged with delayed kinetics but nonetheless completely prevented in vivo readministration. We conclude that there are distinct roles for TLR9 and MyD88 in promoting adaptive immune responses to AAV-mediated gene transfer and that there are redundant MyD88-dependent and MyD88-independent mechanisms that stimulate neutralizing antibody formation against AAV.

Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe Disease

“…67 Additionally, ubiquitous and tissuerestricted promoters used for systemic expression, such as desmin, CMV, or CBA, have a propensity to express in antigen presenting cells, resulting in deleterious innate and adaptive responses that eliminate transduced cells. [68][69][70][71] Therefore, inclusion of an immune tolerance-inducing construct may attenuate the immunogenicity of these vectors. A systemic dose of 5 · 10 13 vg/kg using our dual-vector approach resulted in supraphysiologic levels of enzyme activity in most tissues tested.…”

Differential expression of Toll-like receptor 4 signaling pathway genes in Escherichia coli F18-resistant and – sensitive Meishan piglets

“…The signal can also be conducted through MyD88-independent pathway. The MyD88-independent pathway can generate surprisingly robust IgG antibodies to play an role in antiviral effects (Yamamoto et al 2003a,b, Rogers et al 2015. Identification of drugs that can block or inhibit nodes in the TLR4 signaling pathway are therefore of great clinical interest.…”

Complexity of immune responses to AAV transgene products – Example of factor IX

“…This may in part relate to differences in kinetics and levels of transgene expression. Furthermore, innate immune sensing of the AAV genome by the endosomal DNA receptor TLR9 is a critical signal for the activation of CD8 + T cell responses to the transgene product in skeletal muscle [36, 37]. Using self-complementary vectors increases this signal, while elimination of immune stimulatory CpG motifs (which, in contrast to mammalian DNA, lack methylation in bacterial and viral genomes) reduces CD8 + T cell activation (while, however, having little effect on antibody formation) [36, 38].…”

“…Using self-complementary vectors increases this signal, while elimination of immune stimulatory CpG motifs (which, in contrast to mammalian DNA, lack methylation in bacterial and viral genomes) reduces CD8 + T cell activation (while, however, having little effect on antibody formation) [36, 38]. TLR9-MyD88 signaling has some modulatory effect on Th1 vs Th2 activation and therefore the ratio of different immunoglobulin subclasses that are produced against the AAV capsid and the transgene product, and there are examples of heightened antibody responses when using scAAV vectors [30, 37]. However, the effect on antibody formation overall is modest, as antibodies form even in TLR9 knockout mice [37].…”