DOI: 10.5061/dryad.nk98sf85g
title: Mechanisms of AAV neutralization by human alpha-defensins

description: 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.
description: # Mechanisms of AAV neutralization by human alpha-defensins ## Description
 of the data and file structure Files are provided for each figure as
 follows: **Figure 1A:** 2025_AAVDefensinMechanism_Fig01A_analysis.prism
 Scans of 96-well plates of HeLa cells infected with green fluorescent
 protein-expressing AAV2 were obtained using a multi-mode plate scanner
 from 4 independent replicates. The experimental variable was human
 defensin 5 (HD5). From these images, total well fluorescence was
 enumerated using Fiji by applying a rolling ball background subtraction
 and measuring integrated density limited to threshold. Sample wells were
 normalized to control wells (no HD5), and the normalized data from all
 replicates was analyzed using GraphPad Prism 10.4.1 (.prism). **Figure
 1B:** 2025_AAVDefensinMechanism_Fig01B_analysis.prism Scans of 96-well
 plates of HeLa cells infected with green fluorescent protein-expressing
 AAV2 were obtained using a multi-mode plate scanner from 3 independent
 replicates. The experimental variable was human neutrophil peptide 1
 (HNP1). From these images, total well fluorescence was enumerated using
 Fiji by applying a rolling ball background subtraction and measuring
 integrated density limited to threshold. Sample wells were normalized to
 control wells (no HNP1), and the normalized data from all replicates was
 analyzed using GraphPad Prism 10.4.1 (.prism). **Figure 1C:**
 2025_AAVDefensinMechanism_Fig01C_analysis.prism,
 2025_AAVDefensinMechanism_Fig01C_stoichiometry.prism, and
 2025_AAVDefensinMechanism_Fig01C_stoichiometry.xlsx Sensorgrams were
 obtained from a surface plasmon resonance instrument from 2 independent
 replicates and were visualized in GraphPad Prism 10.4.1 (analysis.prism).
 AAV2 was coupled to the CM5 sensor chip using amine coupling, and 9451
 response units (RUs) were immobilized. The experimental variable was human
 defensin 5 (HD5). RU from these sensorgrams were obtained for each HD5
 concentration at 180 sec and analyzed to determine Bmax, the total number
 of binding sites (stoichiometry.prism). Bmax values were used to calculate
 the stoichiometry of HD5 bound to AAV at equilibrium (.xlxs). **Figure
 1D:** 2025_AAVDefensinMechanism_Fig01D_analysis.prism,
 2025_AAVDefensinMechanism_Fig01D_stoichiometry.prism, and
 2025_AAVDefensinMechanism_Fig01D_stoichiometry.xlsx Sensorgrams were
 obtained from a surface plasmon resonance instrument from 2 independent
 replicates and were visualized in GraphPad Prism 10.4.1 (analysis.prism).
 AAV2 was coupled to the CM5 sensor chip using amine coupling, and 9451
 response units (RUs) were immobilized. The experimental variable was human
 neutrophil peptide 1 (HNP1). RU from these sensorgrams were obtained for
 each HNP1 concentration at 180 sec and analyzed to determine Bmax, the
 total number of binding sites (stoichiometry.prism). Bmax values were used
 to calculate the stoichiometry of HNP1 bound to AAV at equilibrium
 (.xlxs). **Figure 2A:** 2025_AAVDefensinMechanism_Fig02A_analysis.prism
 The experimental variable was human defensin 5 (HD5). For each of 4
 independent replicates, AAV2 bound to HeLa cells at each concentration of
 HD5 was quantified by qPCR against a standard curve. Samples were
 normalized to controls (no HD5), and the normalized data from all
 replicates was analyzed using GraphPad Prism 10.4.1 (.prism). Curves from
 Fig01A and Fig02A were compared using the extra-sum-of-squares F test
 (.prism). **Figure 2B:** 2025_AAVDefensinMechanism_Fig02B_analysis.prism
 The experimental variable was human neutrophil peptide 1 (HNP1). For each
 of 3 independent replicates, AAV2 bound to HeLa cells at each
 concentration of HNP1 was quantified by qPCR against a standard curve.
 Samples were normalized to controls (no HNP1), and the normalized data
 from all replicates was analyzed using GraphPad Prism 10.4.1 (.prism).
 **Figure 2C:** 2025_AAVDefensinMechanism_Fig02C_analysis.prism Scans of
 96-well plates of HeLa cells infected with green fluorescent
 protein-expressing AAV2 were obtained using a multi-mode plate scanner
 from 4 independent replicates. The experimental variable was human
 defensin 5 (HD5). From these images, total well fluorescence was
 enumerated using Fiji by applying a rolling ball background subtraction
 and measuring integrated density limited to threshold. Sample wells were
 normalized to control wells (no HD5), and the normalized data from all
 replicates was analyzed using GraphPad Prism 10.4.1 (.prism). Curves from
 Fig01A and Fig02C were compared using the extra-sum-of-squares F test
 (.prism). **Figure 2D:** 2025_AAVDefensinMechanism_Fig02D_analysis.prism
 Scans of 96-well plates of HeLa cells infected with green fluorescent
 protein-expressing AAV2 were obtained using a multi-mode plate scanner
 from 4 independent replicates. The experimental variable was human
 neutrophil peptide 1 (HNP1). From these images, total well fluorescence
 was enumerated using Fiji by applying a rolling ball background
 subtraction and measuring integrated density limited to threshold. Sample
 wells were normalized to control wells (no HNP1), and the normalized data
 from all replicates was analyzed using GraphPad Prism 10.4.1 (.prism).
 Curves from Fig01B and Fig02D were compared using the extra-sum-of-squares
 F test (.prism). **Figure 3B:**
 2025_AAVDefensinMechanism_Fig03B_analysis.prism Scans of immunoblots of
 AAV2 immunoprecipitated with the A1 and A20 antibodies and probed
 sequentially with the B1 antibody and with goat anti-mouse Alexa Fluor
 488-conjugated secondary antibody were obtained using a multi-mode plate
 scanner from 3 independent replicates. Experimental variables were no
 defensin (ND), human defensin 5 (HD5), and a form of HD5 with aminobutyric
 acid substituted for the six cysteine residues in the linear sequence of
 HD5 (ABU). The background subtracted volume of the VP3 band for each
 sample from each blot was quantified using AzureSpot Pro (v2.1.097)
 software. The ratios of these volumes for the A1 immunoprecipitated
 samples divided by the A20 immunoprecipitated samples from all replicates
 were analyzed using GraphPad Prism 10.4.1 (.prism). **Figure 3D:**
 2025_AAVDefensinMechanism_Fig03D_analysis.prism Scans of immunoblots of
 AAV2 immunoprecipitated with the A1 and A20 antibodies and probed
 sequentially with the B1 antibody and with goat anti-mouse Alexa Fluor
 488-conjugated secondary antibody were obtained using a multi-mode plate
 scanner from 3 independent replicates. Experimental variables were no
 defensin (ND) and human neutrophil peptide 1 (HNP1). The background
 subtracted volume of the VP3 band for each sample from each blot was
 quantified using AzureSpot Pro (v2.1.097) software. The ratios of these
 volumes for the A1 immunoprecipitated samples divided by the A20
 immunoprecipitated samples from all replicates were analyzed using
 GraphPad Prism 10.4.1 (.prism). **Figure 3F:**
 2025_AAVDefensinMechanism_Fig03F_analysis.prism Scans of immunoblots of
 AAV2 immunoprecipitated with the A1 and A20 antibodies and probed
 sequentially with the B1 antibody and with goat anti-mouse Alexa Fluor
 488-conjugated secondary antibody were obtained using a multi-mode plate
 scanner from 3 independent replicates. Experimental variables were no
 defensin (ND) and human defensin 5 (HD5). The background subtracted volume
 of the VP3 band for each sample from each blot was quantified using
 AzureSpot Pro (v2.1.097) software. The ratios of these volumes for the A1
 immunoprecipitated samples divided by the A20 immunoprecipitated samples
 from all replicates were analyzed using GraphPad Prism 10.4.1 (.prism).
 **Figure 3H:** 2025_AAVDefensinMechanism_Fig03H_analysis.prism Scans of
 immunoblots of AAV2 immunoprecipitated with the A1 and A20 antibodies and
 probed sequentially with the B1 antibody and with goat anti-mouse Alexa
 Fluor 488-conjugated secondary antibody were obtained using a multi-mode
 plate scanner from 3 independent replicates. Experimental variables were
 no defensin (ND) and human neutrophil peptide 1 (HNP1). The background
 subtracted volume of the VP3 band for each sample from each blot was
 quantified using AzureSpot Pro (v2.1.097) software. The ratios of these
 volumes for the A1 immunoprecipitated samples divided by the A20
 immunoprecipitated samples from all replicates were analyzed using
 GraphPad Prism 10.4.1 (.prism). **Figure 3K:**
 2025_AAVDefensinMechanism_Fig03K_analysis.prism Scans of 96-well plates of
 HeLa cells infected with green fluorescent protein-expressing AAV2 were
 obtained using a multi-mode plate scanner from 3 independent replicates.
 The experimental variables were no defensin (ND), human defensin 5 (HD5),
 a form of HD5 with aminobutyric acid substituted for the six cysteine
 residues in the linear sequence of HD5 (ABU), and human neutrophil peptide
 1 (HNP1). From these images, total well fluorescence was enumerated using
 Fiji by applying a rolling ball background subtraction and measuring
 integrated density limited to threshold. Sample wells were normalized to
 control wells (ND), and the normalized data from all replicates was
 analyzed using GraphPad Prism 10.4.1 (.prism). **Figure 4A:**
 2025_AAVDefensinMechanism_Fig04A_analysis.prism *Infection data:* Scans of
 96-well plates of HeLa cells infected with green fluorescent
 protein-expressing AAV6 were obtained using a multi-mode plate scanner
 from 4 independent replicates. The experimental variable was human
 defensin 5 (HD5). From these images, total well fluorescence was
 enumerated using Fiji by applying a rolling ball background subtraction
 and measuring integrated density limited to threshold. Sample wells were
 normalized to control wells (no HD5), and the normalized data from all
 replicates was analyzed using GraphPad Prism 10.4.1 (.prism). *Binding
 data:* The experimental variable was human defensin 5 (HD5). For each of 4
 independent replicates, AAV6 bound to HeLa cells at each concentration of
 HD5 was quantified by qPCR against a standard curve. Samples were
 normalized to controls (no HD5), and the normalized data from all
 replicates was analyzed using GraphPad Prism 10.4.1 (.prism). **Figure
 4B:** 2025_AAVDefensinMechanism_Fig04B_analysis.prism *Infection data:*
 Scans of 96-well plates of HeLa cells infected with green fluorescent
 protein-expressing AAV6 were obtained using a multi-mode plate scanner
 from 3 independent replicates. The experimental variable was human
 neutrophil peptide 1 (HNP1). From these images, total well fluorescence
 was enumerated using Fiji by applying a rolling ball background
 subtraction and measuring integrated density limited to threshold. Sample
 wells were normalized to control wells (no HNP1), and the normalized data
 from all replicates was analyzed using GraphPad Prism 10.4.1 (.prism).
 *Binding data:* The experimental variable was human neutrophil peptide 1
 (HNP1). For each of 3 independent replicates, AAV6 bound to HeLa cells at
 each concentration of HNP1 was quantified by qPCR against a standard
 curve. Samples were normalized to controls (no HNP1), and the normalized
 data from all replicates was analyzed using GraphPad Prism 10.4.1
 (.prism). **Figure 4C:** 2025_AAVDefensinMechanism_Fig04C_analysis.prism
 The experimental variables were the ADK1a antibody and wheat germ
 agglutinin (WGA). For each of 6 independent replicates, AAV6 bound to HeLa
 cells in each condition was quantified by qPCR against a standard curve.
 Samples were normalized to controls without inhibitor (None), and the
 normalized data from all replicates was analyzed using GraphPad Prism
 10.4.1 (.prism). **Figure 4F:**
 2025_AAVDefensinMechanism_Fig04F_analysis.prism Scans of immunoblots of
 AAV6 immunoprecipitated with the A1 and ADK1a antibodies and probed
 sequentially with the B1 antibody and with goat anti-mouse Alexa Fluor
 488-conjugated secondary antibody were obtained using a multi-mode plate
 scanner from 4 independent replicates. Experimental variables were no
 defensin (ND), human defensin 5 (HD5), and human neutrophil peptide 1
 (HNP1). The background subtracted volume of the VP3 band for each sample
 from each blot was quantified using AzureSpot Pro (v2.1.097) software. The
 ratios of these volumes for the A1 immunoprecipitated samples divided by
 the ADK1a immunoprecipitated samples were calculated. The A1/ADK1a ratios
 for HD5 and HNP1 samples were normalized to the ratio for the ND control.
 Data from all replicates were analyzed using GraphPad Prism 10.4.1
 (.prism). **Figure 4G:** 2025_AAVDefensinMechanism_Fig04G_analysis.prism
 Scans of 96-well plates of HeLa cells infected with green fluorescent
 protein-expressing AAV6 were obtained using a multi-mode plate scanner
 from 4 independent replicates. The experimental variables were no defensin
 (ND), human defensin 5 (HD5), and human neutrophil peptide 1 (HNP1). From
 these images, total well fluorescence was enumerated using Fiji by
 applying a rolling ball background subtraction and measuring integrated
 density limited to threshold. Sample wells were normalized to control
 wells (ND), and the normalized data from all replicates was analyzed using
 GraphPad Prism 10.4.1 (.prism). **Figure 5C:**
 2025_AAVDefensinMechanism_Fig05C_analysis.prism Peak values corresponding
 to the melting temperature (Tm) of AAV2 were derived from thermograms for
 3 independent replicates. The experimental variables were pH, no defensin
 (ND), human defensin 5 (HD5), and human neutrophil peptide 1 (HNP1). Data
 from all replicates were analyzed using GraphPad Prism 10.4.1 (.prism).
 **Figures 6A, 6B, 6E, and 6F:**
 2025_AAVDefensinMechanism_Fig06A_and_Fig06B_analysis.prism,
 2025_AAVDefensinMechanism_Fig06E_analysis.prism, and
 2025_AAVDefensinMechanism_Fig06F_analysis.prism DyLight 488 intensity for
 HeLa cells infected with DyLight 488-labeled AAV2 was measured from
 micrographs using CellProfiler. The experimental variables were time
 post-infection (p.i.), human defensin 5 (HD5), and human neutrophil
 peptide 1 (HNP1). Control cells were infected in the absence of defensin
 (No defensin, ND). A minimum of 35 cells were analyzed for each condition
 for each of 4 independent replicates. Data from all replicates were
 analyzed using GraphPad Prism 10.4.1 (.prism). For Figures 6A and 6B, the
 fraction of total DyLight 488 signal that was located in the nucleus was
 calculated for each cell. For Figure 6E, the ratio of DyLight 488 signal
 in the nucleus compared to the perinuclear space was calculated for each
 cell, and the log of this ratio was analyzed. For Figure 6F, the total
 DyLight signal is graphed for each cell.