Adeno-associated virus capsid antigen presentation is dependent on endosomal escape

Abstract

Adeno-associated virus (AAV) vectors are attractive for gene delivery-based therapeutics, but data from recent clinical trials have indicated that AAV capsids induce a cytotoxic T lymphocyte (CTL) response that eliminates transduced cells. In this study, we used traditional pharmacological agents and AAV mutants to elucidate the pathway of capsid cross-presentation in AAV-permissive cells. Endosomal acidification inhibitors blocked AAV2 antigen presentation by over 90%, while proteasome inhibitors completely abrogated antigen presentation. Using mutant viruses that are defective for nuclear entry, we observed a 90% decrease in capsid antigen presentation. Different antigen presentation efficiencies were achieved by selectively mutating virion nuclear localization signals. Low antigen presentation was demonstrated with basic region 1 (BR1) mutants, despite relatively high transduction efficiency, whereas there was no difference in antigen presentation between BR2 and BR3 mutants defective for transduction, as compared with wild-type AAV2. These results suggest that effective AAV2 capsid antigen presentation is dependent on AAV virion escape from the endosome/lysosome for antigen degradation by proteasomes, but is independent of nuclear uncoating. These results should facilitate the design of effective strategies to evade capsid-specific CTL-mediated elimination of AAV-transduced target cells in future clinical trials.

Targeting human dendritic cells via DEC-205 using PLGA nanoparticles leads to enhanced cross-presentation of a melanoma-associated antigen

“…This approach is directly analogous to that taken by many pathogens, including viruses, such as adenovirus, reovirus, and papillomavirus, that effect endosome disruption in order to gain access to the cytosol for purposes of infection. [45][46][47][48] Here we have shown that surface modification of Agloaded NPs to direct them to DCs affects the outcome of the immune response towards the delivered antigen. Several 4,8,19 Furthermore, the concept of targeting DEC-205 via particulate delivery to DCs has been introduced in mice, with Kwon et al using pHresponsive microparticles surface-conjugated with the anti-DEC-205 monoclonal antibody to demonstrate that targeting DEC-205 enhances particle uptake and Ag presentation, 49 as well as our groups' demonstration that the density of DEC-205 on the NP surface is crucial to controlling murine DC antigenicity.…”

Recombinant Adeno-Associated Virus Utilizes Host Cell Nuclear Import Machinery To Enter the Nucleus

“…These virions likely become degraded by proteases or are subjected to proteasomal degredation and antigen presentation by host cells (29). Current opinion suggests that if the barrier to nuclear entry were to be overcome, more viruses could translocate to the nucleus for subsequent uncoating and transgene expression.…”

“…We therefore investigated whether the codistribution of rAAV2 and importin-␤ was dependent on later trafficking events that could involve escape from the endosome; we acquired 2D images of cells in the z-plane harboring the center of the nucleus to represent the center of the cell. Endosomal escape has been shown to be inhibited by pharmacological agents that block vesicle acidification (16,29). We therefore utilized chloroquine, a small molecule that blocks the acidification of endosomes, to inhibit later trafficking events and endosomal escape of rAAV2.…”

“…Studies into the intracellular trafficking of rAAV2 have revealed a common pattern: while the majority of particles traffic to a perinuclear region associated with the MTOC, most of these particles remain distal to the nucleus and never translocate (4,5,28). These particles are eventually degraded by host proteasomes and are likely presented as antigens on the cell surface (29). If this barrier to nuclear entry could be overcome, two benefits could be achieved.…”

Syntaxin 5-Dependent Retrograde Transport to the trans -Golgi Network Is Required for Adeno-Associated Virus Transduction

“…Acidification of these carriers triggers a conformational change in the AAV capsid and exposes the N-terminal domain of the minor capsid protein VP1 on the capsid surface (13). VP1 u contains a phospholipase A2 (PLA2) domain that mediates the egress of AAV capsids into the cytoplasm (11,64,65). From the cytoplasm, intact capsids are then translocated across the nuclear envelope to deliver their DNA payload to the nucleus HeLa cells were incubated for 1 h on ice with AAV2-Luc, washed, and transferred to 37°C to trigger infection.…”