Abstract
Background
Recombinant adeno-associated virus vectors based on serotype 2 (AAV-2) have become leading vehicles for gene therapy. Most humans in the general population have anti-AAV-2 antibodies as a result of naturally acquired infections. Pre-existing immunity to AAV-2 might affect the functional and safety consequences of AAV-2 vector-mediated gene transfer in clinical applications.
Methods
An enzyme-linked immunosorbent assay (ELISA) method was developed using microwell plates coated with intact particles of recombinant AAV-2 vectors, and horseradish peroxidase-conjugated anti-human immunoglobulin G (HRP-IgG). Neutralizing antibody titres were analysed by assessing the ability of serum antibody to inhibit transduction into HEK293 cells of AAV vectors that express β-galactosidase.
Results
Anti-AAV-2 antibodies were detected by ELISA in two of 20 healthy subjects. The positivity criterion (optical density >0.5) in ELISA corresponded to the cut-off value (320-fold dilution of serum) in the AAV-2 neutralization assay. Influences of interfering substances were not observed.
Conclusion
This ELISA method may be useful for rapid screening of anti-AAV-2 neutralizing antibodies in candidates for gene therapy.
Introduction
Adeno-associated virus (AAV) is a small single-stranded DNA virus within the parvovirus family. 1,2 Among more than 100 genotypes of primate AAV, serotype 2 (AAV-2) is the most studied and was the first to be engineered for vector development. Recombinant AAV-2 vectors efficiently transduce both dividing and non-dividing cells and provide long-term gene expression without significant toxicity. Growing numbers of clinical trials have been conducted using AAV-2 vectors to combat various diseases. However, one major problem is the high prevalence of anti-AAV-2 antibodies in the human population. More than 90% of adults demonstrate antibodies that cross-react with one or more AAV serotypes, although markedly fewer (18–32%) show neutralizing antibodies (nAb). 3,4 Pre-existing immunity to AAV-2 may block transduction and intensify the innate response to vector administration, leading to a poor outcome of gene therapy. Thus, measurement of the anti-AAV-2 nAb titre is necessary.
Methods
Recombinant AAV-2 vectors were produced by the triple transduction method as described previously. 5 In brief, HEK293 cells were transfected with the following three plasmids: pAAV2-Rep/vp (containing the AAV-2 rep and cap genes), pAd (containing the adenovirus genome) and pW1 (containing the β-galactosidase-expression cassette). After three days of incubation, the transfected cells were frozen and thawed, and the recombinant AAV-2 vector particles that were released were purified by two sequential CsCl density gradient centrifugations.
Serum samples from healthy adults were purchased from Advanced BioServices LLC, (Reseda, CA, USA). AAV-specific antibodies were detected using an enzyme-linked immunosorbent assay (ELISA). Ninety-six-well microtitre plates (Invitrogen, Carlsbad, CA, USA) were coated with 0.5 μg (1.4 × 108 vector genomes [vg]) of AAV-2 vector particles per well. After blocking with 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), the plates were washed with 2% sucrose. Serum samples diluted at 1:1000 with PBS/0.1% BSA were added to each well (100 μL/well). The plates were incubated for 1 h at room temperature (RT) and washed three times with PBS/0.05% Tween 20. A solution containing 1 μg/mL horseradish peroxidase-conjugated anti-human immunoglobulin G (HRP-IgG; self-prepared using a heterobifunctional reagent) was added to each well (100 μL/well). The plates were incubated for 1 h at RT and washed three times with washing buffer. Colour was developed by adding 100 μL 3,3′,5,5′-tetramethylbenzidine/urea hydrogen peroxide (Neogen, Lexington, KY, USA) and incubating the plates for 30 min at RT. Colour development was stopped by adding 1 mol/L sulphuric acid (100 μL/well), and the optical density (OD) was measured at 450/640 nm. The optimum reaction conditions were determined in the following ranges: 0.5 × 108 to 3 × 108 vg of AAV-2 vector particles per well, 10- to 5000-fold dilution of the specimen and 0.01–0.4 μg/mL concentration of HRP-IgG. The effects of bilirubin, haemoglobin and chyle as endogenous interference materials were studied. Samples including these materials at 500 mg/L, 5000 mg/L and 3000°, respectively, were mixed with sera at a volume ratio of 1:9 and analysed by ELISA.
For the nAb assay, sera were continuously diluted two-fold with Dulbecco's Modified Eagle's Medium and Harn's F-12 Nutrient Mixture (DMEM/F12, Invitrogen) 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin (Invitrogen) and 100 μg/mL streptomycin (Invitrogen). AAV-2 vectors were diluted with 10 mmol/L HEPES/130 mmol/L NaCl (pH 8.0) up to 1.4 × 108 vg/μL, and optimum transduction was obtained with 1.4 × 1010 vg/μL. Ten microlitres of diluted serum were added to 5 μL diluted AAV-2 vector, and the mixture was incubated for 1 h at RT. The mixture was then added to 96-well plates containing confluent HEK293 cells. Two days after transduction, β-galactosidase activity was measured with a β-galactosidase assay kit (Invitrogen). The titre of nAb was defined as the highest dilution of serum that showed <50% of the β-galactosidase activity of the negative control.
Results and discussion
In the ELISA method developed in this study, AAV-2 vector particles immobilized on plates captured AAV-2-specific antibodies. Using whole vector particles as antigens without degradation, antibodies that are more specific AAV-2, including nAb, can be assessed. Intra-assay precision was determined by repeatedly (n = 8) measuring four kinds of serum with different nAb titres. For three samples whose nAb titres were ×640, ×80 and ×80, the coefficient of variation (CV) of the OD was 2.5%, 4.3% and 1.8%, respectively. For the fourth sample, with ×10 titre, the average OD was 0.012 and the CV was 8.5%. These data confirm the good precision of the assay. The effects of interference materials were <10%, suggesting that the method had good specificity. All reagents, including AAV particles, were stable after storage for 13 months at 4°C, and sensitivity of the ELISA was maintained at 95% with fresh reagents.
The nAb titres for 20 healthy donor samples varied from ×10 to ×640, and 13 samples had a titre less than ×20. Using the ELISA method, the absorbance values of the samples varied from 0.012 to 0.752, and 15 samples showed absorbance of less than 0.08 (Table 1). In our study, a nAb titre higher than × 32 corresponded to an OD greater than 0.5 in the ELISA. If these values were used as cut-off points, two of 20 samples found to be positive in both the nAb assay and the ELISA. Further studies on more samples were necessary to validate the cut-off values in different populations.
Correlation between a neutralizing antibody assay (nAb) and the ELISA method
OD, optical density; ELISA, enzyme-linked immunosorbent assay; nAb, neutralizing antibodies
AAV-2 antibody in 20 sera from healthy individuals was analysed by ELISA and a neutralizing antibody assay. The measured values were expressed as OD or titre. The cut-off value for the neutralizing antibody assay was provisionally fixed at ×320. The criterion for positivity for the ELISA method was considered greater than OD 0.5
Conclusion
We have developed a simple and convenient ELISA method for detecting serum anti-AAV-2 antibodies. Antibody titres assessed by this method show good correlation with nAb titres obtained in a cell transduction assay, suggesting that this ELISA may be useful for the rapid screening of nAbs in candidates for gene therapy.
DECLARATIONS