Prevalence of Adeno-Associated Virus 3 Capsid Binding and Neutralizing Antibodies in Healthy and Hemophilia B Individuals from India

Abstract

Adeno-associated virus (AAV) vector-based gene therapy offers a new treatment option for individuals with hemophilia. Pre-existing anti-AAV antibodies significantly impact the use of AAV vectors. Even relatively low titers of AAV neutralizing antibodies (NAb) from natural AAV infections against the capsid have been shown to inhibit the transduction of intravenously administered AAV in animal models and were associated with limited efficacy in human trials. This is important for determining the primary eligibility of patients for AAV vector-based gene therapy clinical trials. Current techniques to screen AAV antibodies include AAV capsid enzyme-linked immunosorbent assay (ELISA) for total antibodies and a transduction inhibition assay (TIA) for NAb. This study developed and screened total capsid binding anti-AAV3 antibodies by using ELISA and determined NAb levels by TIA using mCherry flow cytometry in healthy individuals with hemophilia B in India. One hundred and forty-three apparently healthy controls and 92 individuals with hemophilia B were screened. The prevalence of total and NAb in healthy controls was 79.7% and 65%, respectively; the prevalence of total and NAb in patients with hemophilia B for AAV3 was 92.4% and 91.3%, respectively.

Introduction

Adeno-associated virus (AAV) serotype 1 was first identified as a contaminant in Adenovirus cultures and belongs to the Parvoviridae family, genus Dependovirus. ¹ Viruses in this genus require a helper virus for active replication. In the absence of a helper virus, AAV establishes a latent infection by integrating into the host genome or remaining in the episomal form. AAV is classified into 13 serotypes that differ in their structures of capsids.^2
–4 Their lack of pathogenicity, the persistent expression of the transgene, and the availability of many serotypes have made AAV an important gene therapy vector.⁵ AAV serotypes also exhibit diverse tissue tropism, which can deliver the transgene to specific tissue types.^6,7 Various serotypes of AAV have been used in gene therapy.^8,9 However, the presence of anti-AAV antibodies due to natural infection or significant reactivity between serotypes against the capsid antigen is a significant obstacle in using AAV as a gene therapy vector.^10,11

The humoral immune response against AAV is classified into neutralizing and binding antibodies.¹² Studies have shown that very low titers of neutralizing antibodies (NAb) can neutralize the vector and reduce the efficiency of the gene expression of the vector.^13,14 Binding antibodies flag the virus but do not neutralize them.¹⁵ The prevalence of NAb to the different serotypes of AAV varies in different populations.¹⁶ Hence, serotype-specific screening of AAV antibodies is very important for the effective use of AAV as a vector for gene therapy for each application. There is also a challenge in developing practical and reproducible technologies for determining the titers of pre-existing AAV antibodies with assays that have good sensitivity and specificity. Widely used current techniques for the detection of anti-AAV antibodies are capsid enzyme-linked immunosorbent assay (ELISA) and transduction inhibition assay (TIA).^16
–18 The TIA detects neutralizing antibody titer, whereas capsid ELISA detects total antibodies (both neutralizing and non-neutralizing or capsid binding). The objective of this study was to screen for AAV3 total antibodies by using capsid ELISA and for NAb by using TIA by flow cytometry and compare the two methods since this information would be critical before initiating a clinical trial with AAV3 vectors for patients with hemophilia B in India.¹⁹

Materials and Methods

Serum samples

This cross-sectional study was conducted by using 143 serum samples collected from apparently healthy individuals and 92 individuals with hemophilia B after obtaining approval from the Christian Medical College (CMC, Vellore, India) IRB. The 92 individuals with hemophilia B attended a medical camp conducted by the Department of Hematology at CMC, in Tamil Nadu, South India. The 143 healthy individuals were health volunteers and individuals whose serum samples were being screened for preoperative blood-borne viruses. The preoperative samples were recruited after anonymously delinking all patient information except age and gender. All blood samples from hemophilia B and staff members of CMC were collected after obtaining signed informed consent to participate in this study. Of the 143 samples collected from apparently healthy individuals, 82 individuals were older than 18 years, 35 were 5–17 years old, and 26 were 3–5 years old. All patients with hemophilia were ≥18 years old.

AAV reporter vector

AAV3 vectors used for this study carried the gene coding for mCherry driven by the CMV promoter. The AAV3 was produced by using the triple-plasmids-transfection method and purified by using Iodixanol gradient ultra-centrifugation. The vector was procured from PackGene Biotech, LLC (Worcester, MA).

Cell culture

COS-7 cells were procured from ATCC (ATCC^® CRL-1651^™); grown in Dulbecco's modified Eagle's high-glucose medium supplemented with 10% fetal bovine serum (FBS), sodium pyruvate; and used for AAV3 TIA. All cells were maintained in a humidified 37°C incubator with 5% CO₂.

NAb titer assay

COS-7 cells were seeded at a concentration of 3 × 10⁴ cells/well in 48-well plates. Serum samples were diluted by using Iscove's modified Dulbecco's media (IMDM) without FBS. Transduction for AAV3 TIA was done by using a multiplicity of infection (MOI) of 10⁴ vgs. The transduction efficiency of the AAV3 vector was assessed after performing twofold serial dilutions of the vector at different MOIs (20,000 to 625 MOI). An MOI of 10⁴ AAV3 vgs was chosen for this in vitro transduction assay, as this MOI consistently produced >50% transduction efficiency (data not shown). The AAV3 vector at this MOI was added to varying serum dilutions in serum-free IMDM and incubated for 1 h at room temperature (RT). The serum vector complex was added to COS-7 cells in a 48-well plate after washing with 1 × phosphate buffered saline (PBS) to remove FBS and incubated for 2 h at 37°C. Then, serum-free IMDM was replaced with complete medium (IMDM with 10% FBS). After 48 h, the cells were trypsinized and mCherry expression was assessed by using flow cytometry. All samples were screened for NAb at a dilution of 1 in 5 and 1 in 10 in triplicate. Dilution of serum samples was performed in a transduction cocktail (serum+vector+IMDM), with the final concentration of serum being 1 in 5 and 1 in 10 in the transduction cocktail. A20 antibody (an anti-AAV2 antibody that recognizes AAV3; Progen, Heidelberg, Germany) was included at a concentration of 15 ng/well in all the assay plates that served as a positive control. The NAb titer of the sample was calculated as the highest final dilution of the serum, where more than 50% of the cells did not express mCherry compared with the no-serum control. COS-7 cells incubated with serum without AAV3 vector was used as the negative control. COS-7 cells incubated with AAV3 vector without antibodies were used as the no-serum control. All sample dilutions and controls were tested in triplicate.

Enzyme-linked immunosorbent assay

AAV3 empty capsid was coated onto Nunc MaxiSorp flat-bottom plates (Cat. No. 44-2404-2; Thermo Fischer Scientific, MA) at a concentration of 1 × 10⁸ VG/well and incubated overnight at 4°C. The plates are blocked with 3% non-fat dry milk. Samples were diluted 1:40 in 1 × PBS to reduce background and used for screening at this dilution. Fifty microliters of serum were added per well and incubated for 2 h at 37°C. A positive control was included in all assay runs. All serum samples were tested in duplicate. Anti-human IgG HRP conjugate (Cat. No. PA1-28587; Thermo Fischer Scientific, IL) at a concentration of 1:50,000 was added and incubated for 1 h at 37°C. TMB substrate (KPL Seracare) was added and incubated at RT for 30 min, and the reactions were stopped by adding 1 N H₂SO₄. The optical density of the wells was read within 10 min at 450 and 630 nm. Washing between each step was done three times by using PBS with 0.05% Tween 20.

ELISA cutoff

To determine a cutoff for capsid ELISA, all apparently healthy individuals' samples with a TIA titer of 5 or less were considered negative. The geometric mean optical density (OD) of these TIA negative samples was used as a constant to calculate cutoff. A negative control was included in all the ELISA plates. The cutoff used to determine positivity in the ELISA runs was the sum of the mean of negative controls and the constant. $\begin{matrix} E L I S A c u t o f f = m e a n o f t h e n e g a t i v e c o n t r o l \\ (s a m p l e b l a n k) + C O N S T A N T \end{matrix}$

Assay performance

Due to a lack of human anti-AAV3 antibody-positive controls, the AAV3 capsid ELISA method was validated by determining the limit of detection, linearity, and sensitivity using A20 antibody (Progen). A twofold serial dilution of the A20 antibody was used to generate the standard curve. The A20 antibody was diluted in PBS, tested in triplicate on 3 different days, and the mean absorbance was used to determine performance characteristics. Mouse secondary antibody conjugated with HRP was used to detect commercial antibodies with TMB substrate (KPL Seracare). Inter- and intra-assay variation of this assay was assessed by testing a sample in quadruplicate on 3 different days.

Statistical analysis

p-Value <0.05 was considered statistically significant.

Results

ELISA performance

The intra-assay variation was determined by using four different samples tested in quadruplicate on 3 different days. The average coefficient of variations (CV) of the OD obtained was 4.8%, 1.7%, 8%, and 1.2%. The inter-assay CV determined by using a sample tested on 3 different days in quadruplicate was 7.1%. The standard curves generated for AAV3 capsid binding ELISA to assess linearity using commercial antibodies' dilutions are shown in Fig. 1. The sensitivity of the AAV3 capsid ELISA using commercial antibodies was 39 ng/mL.

Figure 1.

Standard curve obtained for AAV3 capsid ELISA using commercially available AAV serotypes 2 antibodies A20. Different dilutions of A20 (cross-reacting to AAV serotype 3) antibodies were prepared for standardization of capsid ELISA assays for AAV3. AAV, adeno-associated virus; ELISA, enzyme-linked immunosorbent assay.

ELISA cutoff

Samples with a titer ≤5 by TIA were considered negative. The geometric mean OD was used as a constant for cutoff determination for ELISA runs. Of the 143 control samples screened for NAb, 50 samples had a titer of 5 or below. The geometric mean OD of AAV3 capsid ELISA in samples with titer ≤5 was 0.7. The AAV3 capsid ELISA OD value of 0.7 was, thus, used as a constant for calculating the cutoff for each run.

Total-binding antibody

The AAV capsid-binding antibody prevalence calculated by using the cutoff in the control and hemophilia B patient groups was 79.7% and 92.4%, respectively (Table 1). In the pediatric age group, 65.6%, and in the adult age group, 90.2% of the individuals were positive for anti-AAV3 total antibodies among healthy individuals. The age-stratified AAV3 antibody prevalence in apparently healthy and hemophilia B individuals showed an increase in prevalence as age progressed (Table 2). The mean total-binding antibody of TIA-negative and -positive samples was 0.9 and 1.6, respectively. There is, thus, a significant difference between the means of AAV3 capsid ELISA OD values in the TIA-negative and -positive samples (p < 0.0001).

Table 1.

Adeno-associated virus 3 total-binding antibody and neutralizing antibody prevalence in apparently healthy controls and individuals with hemophilia B

	Healthy Control (%), N = 143	Hemophilia B Individuals (%), N = 92
Capsid ELISA (total-binding antibodies)	114 (79.7)	85 (92.4)
TIA by flow cytometry (NAb)	93 (65)	84 (91.3)

ELISA, enzyme-linked immunosorbent assay; NAb, neutralizing antibodies; TIA, transduction inhibition assay.

Table 2.

Age-stratified prevalence of adeno-associated virus 3 total antibody in apparently healthy controls and hemophilia B individuals in south India using capsid enzyme-linked immunosorbent assay

Age, Years	TAb
Age, Years	Healthy Control	Hemophilia B Individuals
2–5	18/26 (69.2%)	ND
6–17	22/35 (62.9%)	ND
18–25	29/32 (90.6%)	27/31 (87.1%)
26–35	21/23 (91.3%)	29/30 (96.6%)
≥36	24/27 (88.9%)	29/31 (93.5%)

TAb, total antibody.

Neutralizing antibody

The overall percent prevalence of NAb in the apparently healthy control individuals and hemophilia B individuals was 65% and 91.3%, respectively (Table 1). The age-stratified NAb prevalence in healthy control individuals and hemophilia B individuals is shown in Tables 3 and 4. Of the 143 samples from healthy controls screened, 90 (62.9%) were positive at a dilution >1 in 10. Of the 92 hemophilia B individuals screened, 84 (91.3%) were positive at a dilution >1 in 10.

Table 3.

Age-stratified prevalence of adeno-associated virus 3 neutralizing antibody titer in healthy controls

Age Range, Years	N	Median Age	Gender, M/F	NAb Titer				Total Positives >5 (%)
Age Range, Years	N	Median Age	Gender, M/F	<5	5	10	>10	Total Positives >5 (%)
2–5	26	4	20/6	16 (61.5%)	1	0	9 (34.6%)	9 (34.6%)
6–17	35	9	31/4	18 (51.4%)	0	0	17 (48.6%)	17 (48.6%)
18–25	32	23	19/13	4 (12.5%)	3	2	23 (71.9%)	25 (78.1%)
26–35	23	29	15/8	4 (17.4%)	1	1	17 (73.9%)	18 (78.2%)
≥36	27	41	22/5	3 (11.1%)	0	0	24 (88.9%)	24 (88.9%)

Table 4.

Age-stratified prevalence of adeno-associated virus 3 neutralizing antibody titer in individuals with hemophilia B

Age, Years	N	Median Age	Sex, M/F	NAb Titer				Total Positives >5 (%)
Age, Years	N	Median Age	Sex, M/F	<5	5	10	>10	Total Positives >5 (%)
6–17	—		—	—	—	—	—	—
18–25	31	21	30/1	2 (6.5%)	2	1	26 (83.9%)	27 (87.1%)
26–35	30	30	30/0	1 (3.3%)	1	1	27 (90%)	28 (93.3%)
≥36	31	42	31/0	0	2	1	28 (90.3%)	29 (93.5%)

Compared with TIA in the healthy controls and hemophilia B individuals, the capsid ELISA's sensitivity was 95.7% and 94.05%, respectively. The specificity of capsid ELISA compared with TIA in the general population and the hemophilia B patient group was 50% and 25%, respectively (Tables 5 and 6). A scatter plot comparing total/capsid binding antibody OD with TIA is shown in Fig. 2. There is no significant difference between the control group and the hemophilia B group for age groups 18–25 (p = 0.35), 26–35 (p = 0.115), and ≥36 (p = 0.56).

Figure 2.

Scatter plots comparing total anti-AAV3 antibody OD by capsid ELISA and neutralizing antibody by TIA. Samples are considered positive above the defined cutoff (ELISA cutoff by described formula and 50% transduction inhibition cutoff used in TIA for healthy controls and individuals with hemophilia B at 1/5 dilution of serum samples). The top-left quadrant shows samples positive by capsid ELISA and TIA. The top right quadrant shows samples positive by capsid ELISA and negative by TIA. The bottom left quadrant shows samples negative by capsid ELISA and positive by TIA. The bottom right quadrant shows samples negative by capsid ELISA and TIA. (A) Comparison between AAV3 capsid ELISA and TIA at 1/5 dilution of serum in patients with hemophilia B. (B) Comparison between AAV3 capsid ELISA and TIA at 1/5 dilution of serum in apparently healthy individuals. (C) Comparison between AAV3 capsid ELISA and TIA at 1/10 dilution of serum in patients with hemophilia B. (D) Comparison between AAV3 capsid ELISA and TIA at 1/10 dilution of serum in apparently healthy individuals. TIA, transduction inhibition assay.

Table 5.

Adeno-associated virus 3 capsid enzyme-linked immunosorbent assay characteristics in comparison to transduction inhibition assay positivity in healthy controls (n = 143)

	TIA Positive (>Titer 5)	TIA Negative (≤Titer 5)	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)
ELISA positive (≥0.7)	89	25	95.7	50.0	78.07	86.21
ELISA negative (<0.7)	4	25	95.7	50.0	78.07	86.21

PPV, positive predictive value; NPV, negative predictive value.

Table 6.

Adeno-associated virus 3 capsid enzyme-linked immunosorbent assay characteristics in comparison to transduction inhibition assay positivity in individuals with hemophilia B (n = 92)

	TIA Positive (≥Titer 10)	TIA Negative (≤Titer 5)	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)
ELISA positive (≥0.7)	79	6	94.05	25.	92.94	28.57
ELISA negative (<0.7)	5	2	94.05	25.	92.94	28.57

Discussion

In this study, we have established a sensitive in-house capsid-binding ELISA that detects total AAV3 capsid-binding antibodies compared with the TIA, which detects NAb against the AAV3 capsid antigen. The significant advantage of using a capsid ELISA compared with a cell-based NAb assay is that it is less cumbersome, easy to develop, and less variable. Therefore, it is easy to perform in resource-poor countries with minimal expertise. The robustness of this ELISA is good as assessed by inter- and intra-assay variation. This total antibody assay can be developed into a semi-quantitative assay by using purified anti-AAV IgG. The lack of AAV3 antibody standards for ELISA is a significant concern for establishing a cutoff for ELISA and seroprevalence assessment. AAV-mediated gene therapy can be severely hampered due to the presence of even a low titer (≥1 in 5) NAb.²⁰ Since human anti-AAV3 antibody controls were not available, semi-quantitative antibody analysis could not be achieved. This study has considered samples with a titer ≤5 by TIA as negative and used it to determine cutoff (background) for this ELISA.

Moreover, a titer of 5 by TIA is used as the criterion for selecting individuals for gene therapy. We have used AAV3 TIA as the gold standard to compare the performance of this capsid ELISA. ELISA and TIA assessment of AAV3 antibodies shows a significant correlation between assays while using human serum from controls and individuals with hemophilia B. This capsid ELISA has a good sensitivity compared with TIA but low specificity. The low specificity observed in the capsid ELISA of 50% and 25% in the control and hemophilia B groups, respectively, compared with TIA, is probably due to its ability to detect both NAb and non-NAb of AAV3. In this study group, 21.9% and 7.1% of the healthy and hemophilia individuals were negative for NAb but were positive for total antibodies.

Further clinical evaluation of this group of individuals needs to be done to assess their recruitment for gene therapy, as there are studies indicating non-NAb enhancing transduction in cell-based assays.¹⁵ Previous studies comparing total and NAb have varied results.²¹ Different serotypes of AAV elicit varied total to NAb ratio.²² In this study, 78.1% and 92.9% of the healthy and hemophilia individuals, respectively, who had total anti-AAV3 antibody produced NAb.

There are very little data on the seroprevalence of pre-existing AAV3 antibodies worldwide, with no data from India (Table 7). This study provides the first estimate of seroprevalence of AAV3 in India among hemophilia B individuals and apparently healthy individuals, which could significantly impact the design of gene therapy trials in the Indian population. Our data suggest a high prevalence of pre-existing antibodies to AAV3 among individuals with hemophilia B and apparently healthy individuals from India. A similar study in the Chinese population also demonstrated a very high prevalence of 89%.²³ It is well understood that seroprevalence to AAV serotypes varies between different geographic regions, ranging from 3.2% to about 80%.^11,24
–26 We have used mCherry as the reporter gene, which could have contributed to the high prevalence. Different reporter genes may affect NAb detection differently, with the luciferase gene being reported to be more sensitive.²⁷

Table 7.

Adeno-associated virus neutralizing and total antibody prevalence around the globe

Geographical Area	Antibody	AAV1 (%)	AAV2 (%)	AAV3 (%)	AAV5 (%)	AAV6 (%)	AAV8 (%)	AAV9 (%)	References
United States	Nab	—	30	—	18	30	—	—	Halbert et al. ²⁵
France	TAb, Nab	67, 50.5	72, 59	—	40, 3.2	46, 37	38, 19	47, 33.5	Boutin et al. ¹¹
Australia	Nab	30	35	—	—	—	27	—	Calcedo et al. ¹⁶
Europe	Nab	27	35	—	—	—	22	—	Calcedo et al. ¹⁶
Africa	Nab	43	56	—	—	—	31	—	Calcedo et al. ¹⁶
United States	Nab	—	62.5	—	—	—	—	—	Murphy et al. ²⁹
United States	TAb, Nab	—	96, 32	—	—	—	—	—	Chirmule et al. ²¹
Japan	Nab	36.5	35.3	—	37.6	—	32.9	36.5	Mimuro et al. ²⁴
China	Nab	—	96.6	—	40.2	—	82	—	Liu et al. ²⁶
China	Nab	—	92	89	—	—	69	—	Ling et al. ²³

AAV, adeno-associated virus.

Age-stratified analysis in the Indian healthy population reveals a similar trend for total and NAb prevalence, with high prevalence in the age groups above 18. In this study, seroprevalence was 69.2% and 34.6% in the age group of 3–5 for total and NAb, respectively, with ∼90% of the population being seropositive at the age of 36. In the hemophilia B group, the incidence was high in the age group above 18 who are eligible for gene therapy.

We plan to evaluate this ELISA further for cross-reactivity between the AAV serotypes. Of crucial importance is that a robust antibody standard has to be generated for quantitation and screening anti-AAV antibodies against different serotypes in a standardized manner by both ELISA and TIA methods. Importance should be given to establishing a threshold of anti-AAV3 pre-existing antibodies, which decreases the expression of the vector gene in a clinical setting.

Conclusion

We have screened for AAV3 NAb by a TIA using mCherry as the detection system. This assay can be further improved and made cost-effective by using 96-well plates for screening using luciferase as the detection system. The high prevalence of anti-AAV3 total and NAb reported in this study will influence the design of clinical trials in individuals with hemophilia B in India. However, given such a high prevalence, an alternative strategy, recently described by Leborgne et al., might be needed.²⁸

Footnotes

Authors' Contributions

All authors certify that they have participated in the concept, design, analysis, writing, or revision of the article. This article is not published or submitted to any other journal.

Acknowledgments

The authors acknowledge the staff of the Department of Hematology who helped with sample collection, and the patients and their families for being a part of this study.

Author Disclosure

M.A.-M. is a scientific advisor of Voyager Therapeutics, is a member of the Scientific Advisory Board (SAB) for Applied Genetic Technologies Corporation (AGTC), is a consultant for Intima Biosciences, and has a sponsored research agreement with Intima Bioscience and Voyager Therapeutics. She is also a co-founder of StrideBio, and has intellectual property on AAV vectors. Ar.S. is a co-founder of, and holds equity in, Lacerta Therapeutics and Nirvana Therapeutics and is an inventor on several issued patents on recombinant AAV vectors that have been licensed to various gene therapy companies. All authors declare no conflict of interest.

Funding Information

The authors acknowledge the Department of Biotechnology (DBT), Government of India for research funding (Grant No. BT/PR17316/MED/31/326/2015).

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