Mechanisms of AAV neutralization by human alpha-defensins

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.