Recent Advances in Designing Adeno-Associated Virus-Based Vaccines Against Viral Infections

Figure 1. AAV/rAAV structure and production workflow. (A) The particle structure is made of a 22 nm capsid that encapsulates a 4.7 kb single-stranded DNA genome (1). The genome bears two inverted terminal repeats (ITRs) that flank the rep and the cap open reading frames. Gene expression is driven by three promoters (p5, p19, and p40, 2). The rAAV genome can be in an ssDNA form (ssAAV) with a packaging capacity of 4.7 kb (3) or a self-complementary DNA form (scAAV) with a packaging capacity of 2.5 kb (4). (B) The rAAV workflow generally involves a triple transfection of HEK293T cells and incubation to allow viral replication (1). The cultures are centrifuged to harvest the cells and the supernatant (2). The cell pellet is lysed and clarified by centrifugation (3a), and polyethylene glycol (PEG) precipitation of the virus from the supernatant is performed to concentrate the virus (3b). The clarified lysate is used to resuspend the PEG pellet. The lysate is then purified by iodixanol gradient ultracentrifugation and/or chromatography (4) before titration by q-PCR and purity assessment by polyacrylamide gel electrophoresis (PAGE, 5). The purified rAAV is used for in vitro and in vivo studies…

Figure 2. rAAV replication cycle. The rAAV capsid binds to primary receptors on the cell surface (1), facilitating interaction with secondary co-receptors (2) for internalization via clathrin-mediated, caveolin-mediated, or clathrin-independent endocytosis (3). Following endocytosis, rAAV particles escape the early (EEs) or late endosomes (LEs) to the cytosol before targeting the Golgi apparatus (4) or nucleus directly (5). In the event of failure to escape, rAAVs can be degraded by the lysosomes or proteasomes. Once in the nucleus, the rAAV genome is released from the capsid (6). For ssAAVs, the single-stranded DNA genome is converted into double-stranded DNA (7), while the scAAV genome can directly serve as a template for transcription (8a) followed by rep protein synthesis (9). Rep proteins then mediate genome replication (10) with the newly formed double-stranded genome copies serving as the major template for synthesis of cap proteins encoding RNA (8b), followed by rep protein synthesis (11), viral assembly, and release (12) (Created in BioRender.com).

Figure 3. AAV modifications to enhance immunogenicity. (A) Genome modifications. AAVs carrying single-stranded AAV (ssAAV) allow the capsid to act as an adjuvant before ssAAV achieves full antigen expression to boost the vaccine effect (1). Self-complementary AAVs or any genome conformation carrying unmethylated CpG islands are recognizable by Toll-like receptor-9 (TLR-9) receptors, whose signaling leads to pro-inflammatory cytokine response (2). Incorporation of sequences encoding various signal peptides, including MHC-I trafficking signal peptide, IgE/IL-2 secretory domain, and tissue plasminogen activator (tPA), or the T4 fibritin-derived trimeric motif or adjuvants, such as RS09 and ovalbumin, direct antigens to the endoplasmic reticulum (ER), where peptides bind to MHC I for antigen presentation or to improve antigen folding/secretion and/or boost overall immune responses (3). (B) Capsid modification. Capsid-bearing dendritic cell (DC) targeting peptide or S663V point mutation transduces DCs efficiently (1). Capsids with MHC epitopes target antigen-presenting cells (APCs) (2). Overall, all the modifications enhance antibody (Ab) and T-cell responses (created in BioRender.com).