AAP8 Antibody

Definition and Target Specificity

Anti-AAV8 antibodies are monoclonal or chimeric immunoglobulins designed to recognize and neutralize AAV8 viral capsids. These antibodies are pivotal for:

• Quality control of AAV vector preparations (e.g., quantifying intact vs. denatured capsids) .

• Neutralization assays to assess vector efficacy in gene therapy .

• Serology studies to evaluate pre-existing immunity in patients .

Key targets include conformational epitopes on intact AAV8 capsids, with cross-reactivity observed for AAV3, AAV7, AAVrh10, and AAVrh74 .

Key Antibody Clones and Properties

Notes:

• ADK8-h1 is a recombinant chimeric antibody with human Fc regions, minimizing immunogenicity in clinical applications .

• ADK8 neutralizes AAV8 by binding capsid regions critical for cellular entry .

Neutralization Mechanisms

• Dose-dependent inhibition: ADK8-h1 achieves 50% neutralization (EC₅₀) at antibody concentrations as low as 0.2 ng/ml .

• Epitope specificity: Anti-AAV8 antibodies target the VP1/VP2/VP3 capsid proteins, particularly the C-terminal domain critical for capsid assembly .

Clinical Relevance

• Gene therapy safety: In a phase 1 trial, intramuscular AAV8 vectors coding for HIV-neutralizing antibodies (VRC07) showed no significant CD4+ T cell decline or viral load rebound over 3 years .

• Immune responses: Anti-AAV8 antibodies induced high serum titers (>1:21,870) post-administration, persisting for >1 year .

Cross-Reactivity Challenges

Recommendation: Use knockout cell lines (e.g., APP-null) to validate antibody specificity .

Dot Blot Procedure

1. Blocking: 5% milk in PBST, 45 min at RT.

2. Primary antibody: 100 ng/ml anti-AAV8 in blocking buffer (1.5 h incubation).

3. Detection: Chemiluminescent substrate (e.g., Pierce™ ECL) with anti-human IgG-HRP conjugate.

Neutralization Assay

• Procedure: Pre-incubate AAV8-Luc vectors with antibodies (0.2–3,000 ng/ml) for 30 min before infecting HeLa cells.

• Outcome: Luciferase activity reduction correlates with neutralization efficiency (e.g., >90% inhibition at 300 ng/ml ADK8) .

Limitations and Future Directions

• Pre-existing immunity: Anti-AAV8 antibodies in >30% of populations necessitate capsid engineering to evade neutralization .

• Epitope mapping: Structural studies are needed to resolve capsid-antibody interactions at atomic resolution .