Skip to main content
Dryad

Mechanisms of AAV neutralization by human alpha-defensins

Porter, Jessica M.1; Hulce, Kaitlin R.1; Oswald, Mackenzi S.1; Busuttil, Kevin2; Emmanuel, Shanan2; Bennett, Antonette2; McKenna, Robert2; Smith, Jason G.1

Research facility: National Institute of Allergy and Infectious Diseases
Published Jul 14, 2025 on Dryad. https://doi.org/10.5061/dryad.nk98sf85g

Data files

Jul 14, 2025 version files 2.44 MB

Abstract

Antiviral immunity compromises the efficacy of adeno-associated virus (AAV) vectors used for gene therapy. This is well understood for the adaptive immune response. However, innate immune effectors like alpha-defensin antimicrobial peptides also block AAV infection, although their mechanisms of action are unknown. To address this gap in knowledge, we investigated AAV2 and AAV6 neutralization by human neutrophil peptide 1 (HNP1), a myeloid alpha-defensin, and human defensin 5 (HD5), an enteric alpha-defensin. We found that both defensins bind to AAV2 and inhibit infection at low micromolar concentrations, similar to our prior studies of AAV6. While HD5 prevents AAV2 and AAV6 from binding to cells, HNP1 does not. However, AAV2 and AAV6 exposed to HD5 after binding to cells are still neutralized, indicating an additional block to infection. Accordingly, both HD5 and HNP1 inhibit externalization of the VP1 unique domain of both AAV2 and AAV6, which contains a phospholipase A2enzyme required for endosome escape and nuclear localization signals required for nuclear entry. Consequently, both defensins prevent AAV from reaching the nucleus. Disruption of intracellular trafficking of the viral genome to the nucleus is reminiscent of how alpha-defensins neutralize other non-enveloped viruses, suggesting a common mechanism of inhibition. These results will inform the development of vectors capable of overcoming these hurdles to improve the efficiency of gene therapy. AAVs are commonly used as gene therapy vectors due to their broad tropism and lack of disease association; however, host innate immune factors, such as human alpha-defensin antimicrobial peptides, can hinder gene delivery. Although it is becoming increasingly evident that human alpha-defensins can block infection by a wide range of nonenveloped viruses, including AAVs, their mechanism of action remains poorly understood. In this study, we describe for the first time how two types of abundant human alpha-defensins neutralize two AAV serotypes, AAV2 and AAV6. We found that one defensin prevents AAV binding to cells, the first step in infection, while both defensins block a critical later step in AAV entry. Our findings support the emerging idea that defensins use a common strategy to block infection by DNA viruses that replicate in the nucleus. Through understanding how innate immune effectors interact with and impede AAV infection, vectors can be developed to bypass these interventions and allow more efficient gene delivery.