Mapping the Structural Determinants Responsible for Enhanced T Cell Activation to the Immunogenic Adeno-Associated Virus Capsid from Isolate Rhesus 32.33

Abstract

bAvoiding activation of immunity to vector-encoded proteins is critical to the safe and effective use of adeno-associated viral (AAV) vectors for gene therapy. While commonly used serotypes, such as AAV serotypes 1, 2, 7, 8, and 9, are often associated with minimal and/or dysfunctional CD8 ؉ T cell responses in mice, the threshold for immune activation appears to be lower in higher-order species. We have modeled this discrepancy within the mouse by identifying two capsid variants with differential immune activation profiles: AAV serotype 8 (AAV8) and a hybrid between natural rhesus isolates AAVrh32 and AAVrh33 (AAVrh32.33). Here, we aimed to characterize the structural determinants of the AAVrh32.33 capsid that augment cellular immunity to vector-encoded proteins or those of AAV8 that may induce tolerance. We hypothesized that the structural domain responsible for differential immune activation could be mapped to surface-exposed regions of the capsid, such as hypervariable regions (HVRs) I to IX of VP3. To test this, a series of hybrid AAV capsids was constructed by swapping domains between AAV8 and AAVrh32.33. By comparing their ability to generate transgene-specific T cells in vivo versus the stability of transgene expression in the muscle, we confirmed that the functional domain lies within the VP3 portion of the capsid. Our studies were able to exclude the regions of VP3 which are not sufficient for augmenting the cellular immune response, notably, HVRs I, II, and V. We have also identified HVR IV as a region of interest in conferring the efficiency and stability of muscle transduction to AAVrh32.33.

The structure of AAVrh32.33, a novel gene delivery vector

“…6). Significant for this virus, the CD8+ T-cell response mediated by this virus has been mapped to the VP3 region, and based on comparative studies with AAV8, is localized to the VRs, although not VR-I, VR-II, or VR-V (Mays et al, 2013). VR-IV was also observed to be an important muscle transduction determinant for this virus.…”

“…AAV4, a serotype that is one of the most structurally and serologically different from other human and non-human primate AAVs (Gao et al, 2004; Govindasamy et al, 2006) but shares high (~81%) sequence similarity with AAVrh32.33, was found to have a similar low antibody response (Calcedo et al, 2009). Thus AAVrh32.33 (and AAV4) are attractive for development as a gene delivery vectors with the potential to evade pre-existing host antibody responses against AAV capsids (Calcedo et al, 2009; Li et al, 2012; Mays and Wilson, 2009; Mays et al, 2009). AAVrh32.33 is also being evaluated as a genetic vaccine platform, specifically for human immunodeficiency virus type 1 (HIV-1) and type A influenza virus (Lin et al, 2009).…”

“…These studies thus identify AAVrh32.33 as a vector that enables the stimulation of immune responses to expressed transgenes. Interestingly, a comparative study of AAVrh32.33 and AAV8, another non-human primate serotype, showed that the latter induces a minimal CD8+ T-cell response (Mays et al, 2009). The AAV capsid is reported to play a major role in this phenotype (Mays et al, 2013).…”

Strategies to circumvent humoral immunity to adeno-associated viral vectors

“…Early studies indicate that only 2% of the human population carry NAbs against certain rhesus isolates, AAVrh32.33 [16]. However, the latter strain is also highly immunogenic, which could restrict applications in the gene therapy context [44]. It should also be noted that human IgG prevalence as high as 49% accompanied by a lower NAb seroprevalence of 21% has been reported against certain promising rhesus isolates such as AAVrh.10 [45].…”

In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector

“…A wealth of naturally occurring AAV variants have been studied extensively and yielded large descriptive data sets (Gao et al, 2003; Gao et al, 2004; Gao et al, 2005). Forward genetic studies have resulted in important findings (Adachi et al, 2014; Asuri et al, 2012; Bartel et al, 2012; Dalkara et al, 2013; Huttner et al, 2003; Koerber et al, 2008; Li et al, 2008; Lisowski et al, 2014; Maheshri et al, 2006; Mays et al, 2013; Perabo et al, 2003; Shen et al, 2007; Wu et al, 2000) but often remain constrained by the limited tolerance for structural change of the rigid architecture of AAV (Adachi et al, 2014; Huttner et al, 2003; Lochrie et al, 2006; Mays et al, 2013; Shen et al, 2007; Vandenberghe et al, 2009; Wu et al, 2000). Rational AAV design has therefore been difficult in structurally isolating and modulating vector phenotypes while retaining integrity and the desirable aspects of its innate function and biology of the particle.…”