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Prevalence and pharmacological modulation of humoral immunity to AAV vectors in gene transfer to synovial tissue.

Mingozzi F, Chen Y, Edmonson SC, Zhou S, Thurlings RM, Tak PP, High KA, Vervoordeldonk MJ - Gene Ther. (2012)

Bottom Line: This difference was more evident for AAV2, against which higher titers were measured.A drop of NAb titer was observed in a subset of those subjects carrying NAb titers ≤1:1000; however, only in a minority of subjects titers dropped below 1:5.This work provides insights into strategies to overcome the limitation of pre-existing humoral immunity to AAV vectors.

View Article: PubMed Central - PubMed

Affiliation: Center of Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. mingozzi@email.chop.edu

ABSTRACT
Antibodies against adeno-associated viral (AAV) vectors are highly prevalent in humans. Both preclinical and clinical studies showed that antibodies against AAV block transduction even at low titers, particularly when the vector is introduced into the bloodstream. Here we measured the neutralizing antibody (NAb) titer against AAV serotypes 2, 5, 6 and 8 in the serum and matched synovial fluid (SF) from rheumatoid arthritis patients. The titer in the SF was lower than that in the matched plasma samples, indicating a difference in distribution of NAb to AAV depending on the body fluid compartment. This difference was more evident for AAV2, against which higher titers were measured. Of all serotypes, anti-AAV5 antibodies were the least prevalent in both the serum and SF. We next evaluated the impact of B-cell depletion on anti-AAV antibodies in rheumatoid arthritis patients who received one or two courses of the anti-CD20 antibody rituximab as part of their disease management. A drop of NAb titer was observed in a subset of those subjects carrying NAb titers ≤1:1000; however, only in a minority of subjects titers dropped below 1:5. This work provides insights into strategies to overcome the limitation of pre-existing humoral immunity to AAV vectors.

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Related in: MedlinePlus

Comparison of anti-AAV NAb titer determinations using ssAAV-LacZ or scAAV-Luc reporter vectors. The assays shown are a representation of a larger set. A different serum sample from an individual donor was used in each plot. Anti-AAV NAb titers were determined using an in vitro assay in which an ssAAV-LacZ (blue lines) or scAAV-Luc (red lines) reporter transgene vectors were used. (a–c) Anti-AAV2 NAb; (d–f) anti-AAV5; (g–i) anti-AAV6; and (j–l) anti-AAV8 NAb titer determinations. Results are reported as % inhibition of the reporter gene signal (y axis) after incubation of the reporter AAV vector with each given dilution of the serum (x axis). Horizontal lines represent an inhibition of reporter signal of 50%. Titers reported in each graph represent the serum dilution at which the inhibition of the reporter signal fell below 50% blue numbers, titers obtained with the ssAAV-LacZ vector; red numbers, titers obtained with the scAAV-Luc vector. Error bars represent the standard deviation of the average of triplicate readings.
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fig2: Comparison of anti-AAV NAb titer determinations using ssAAV-LacZ or scAAV-Luc reporter vectors. The assays shown are a representation of a larger set. A different serum sample from an individual donor was used in each plot. Anti-AAV NAb titers were determined using an in vitro assay in which an ssAAV-LacZ (blue lines) or scAAV-Luc (red lines) reporter transgene vectors were used. (a–c) Anti-AAV2 NAb; (d–f) anti-AAV5; (g–i) anti-AAV6; and (j–l) anti-AAV8 NAb titer determinations. Results are reported as % inhibition of the reporter gene signal (y axis) after incubation of the reporter AAV vector with each given dilution of the serum (x axis). Horizontal lines represent an inhibition of reporter signal of 50%. Titers reported in each graph represent the serum dilution at which the inhibition of the reporter signal fell below 50% blue numbers, titers obtained with the ssAAV-LacZ vector; red numbers, titers obtained with the scAAV-Luc vector. Error bars represent the standard deviation of the average of triplicate readings.

Mentions: To improve the sensitivity of detection of NAb in vitro in a cell culture-based assay, we switched the AAV transgene expression cassette from a single-stranded genome expressing β-galactosidase (ssAAV-LacZ) to a self-complementary genome20, 21 expressing the luciferase reporter transgene (scAAV-Luc). The higher efficiency of transduction of self-complementary AAV in vitro compared with single-stranded AAV, and the higher sensitivity of detection of luciferase over β-galactosidase, allowed the use of multiplicity of infections 50- to 150-fold lower in the NAb assay. To test the efficiency of the improved assay, we rescreened various serum samples with known NAb titers (all <1:100) using the two assays (Figure 2). As expected, there was no significant difference in the anti-AAV-2 NAb titer resulting from the two assays, reflecting the high efficiency of cell transduction of this AAV serotype in vitro (Figures 2a–c). For AAV5, 6 and 8, much less efficient in transducing cells in vitro, the NAb titers in the serum measured with the ssAAV-LacZ vectors were lower than that with the scAAV-Luc vectors (Figures 2d–l). For these serotypes, the limited sensitivity and the high background noise measured in the LacZ-based assay resulted in a high variability of optical density readings for each dilution (Figure 2). NAb titers (a representative set of a larger number of experiments performed), determined as the serum dilution at which 50% of inhibition of reporter gene expression is measured, are reported in Figure 2 for both the β-galactosidase and the luciferase-based assays. These data indicate that for AAV serotype other than AAV2, the choice of the reporter gene system is a crucial determinant of the sensitivity of the NAb assay.


Prevalence and pharmacological modulation of humoral immunity to AAV vectors in gene transfer to synovial tissue.

Mingozzi F, Chen Y, Edmonson SC, Zhou S, Thurlings RM, Tak PP, High KA, Vervoordeldonk MJ - Gene Ther. (2012)

Comparison of anti-AAV NAb titer determinations using ssAAV-LacZ or scAAV-Luc reporter vectors. The assays shown are a representation of a larger set. A different serum sample from an individual donor was used in each plot. Anti-AAV NAb titers were determined using an in vitro assay in which an ssAAV-LacZ (blue lines) or scAAV-Luc (red lines) reporter transgene vectors were used. (a–c) Anti-AAV2 NAb; (d–f) anti-AAV5; (g–i) anti-AAV6; and (j–l) anti-AAV8 NAb titer determinations. Results are reported as % inhibition of the reporter gene signal (y axis) after incubation of the reporter AAV vector with each given dilution of the serum (x axis). Horizontal lines represent an inhibition of reporter signal of 50%. Titers reported in each graph represent the serum dilution at which the inhibition of the reporter signal fell below 50% blue numbers, titers obtained with the ssAAV-LacZ vector; red numbers, titers obtained with the scAAV-Luc vector. Error bars represent the standard deviation of the average of triplicate readings.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473155&req=5

fig2: Comparison of anti-AAV NAb titer determinations using ssAAV-LacZ or scAAV-Luc reporter vectors. The assays shown are a representation of a larger set. A different serum sample from an individual donor was used in each plot. Anti-AAV NAb titers were determined using an in vitro assay in which an ssAAV-LacZ (blue lines) or scAAV-Luc (red lines) reporter transgene vectors were used. (a–c) Anti-AAV2 NAb; (d–f) anti-AAV5; (g–i) anti-AAV6; and (j–l) anti-AAV8 NAb titer determinations. Results are reported as % inhibition of the reporter gene signal (y axis) after incubation of the reporter AAV vector with each given dilution of the serum (x axis). Horizontal lines represent an inhibition of reporter signal of 50%. Titers reported in each graph represent the serum dilution at which the inhibition of the reporter signal fell below 50% blue numbers, titers obtained with the ssAAV-LacZ vector; red numbers, titers obtained with the scAAV-Luc vector. Error bars represent the standard deviation of the average of triplicate readings.
Mentions: To improve the sensitivity of detection of NAb in vitro in a cell culture-based assay, we switched the AAV transgene expression cassette from a single-stranded genome expressing β-galactosidase (ssAAV-LacZ) to a self-complementary genome20, 21 expressing the luciferase reporter transgene (scAAV-Luc). The higher efficiency of transduction of self-complementary AAV in vitro compared with single-stranded AAV, and the higher sensitivity of detection of luciferase over β-galactosidase, allowed the use of multiplicity of infections 50- to 150-fold lower in the NAb assay. To test the efficiency of the improved assay, we rescreened various serum samples with known NAb titers (all <1:100) using the two assays (Figure 2). As expected, there was no significant difference in the anti-AAV-2 NAb titer resulting from the two assays, reflecting the high efficiency of cell transduction of this AAV serotype in vitro (Figures 2a–c). For AAV5, 6 and 8, much less efficient in transducing cells in vitro, the NAb titers in the serum measured with the ssAAV-LacZ vectors were lower than that with the scAAV-Luc vectors (Figures 2d–l). For these serotypes, the limited sensitivity and the high background noise measured in the LacZ-based assay resulted in a high variability of optical density readings for each dilution (Figure 2). NAb titers (a representative set of a larger number of experiments performed), determined as the serum dilution at which 50% of inhibition of reporter gene expression is measured, are reported in Figure 2 for both the β-galactosidase and the luciferase-based assays. These data indicate that for AAV serotype other than AAV2, the choice of the reporter gene system is a crucial determinant of the sensitivity of the NAb assay.

Bottom Line: This difference was more evident for AAV2, against which higher titers were measured.A drop of NAb titer was observed in a subset of those subjects carrying NAb titers ≤1:1000; however, only in a minority of subjects titers dropped below 1:5.This work provides insights into strategies to overcome the limitation of pre-existing humoral immunity to AAV vectors.

View Article: PubMed Central - PubMed

Affiliation: Center of Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. mingozzi@email.chop.edu

ABSTRACT
Antibodies against adeno-associated viral (AAV) vectors are highly prevalent in humans. Both preclinical and clinical studies showed that antibodies against AAV block transduction even at low titers, particularly when the vector is introduced into the bloodstream. Here we measured the neutralizing antibody (NAb) titer against AAV serotypes 2, 5, 6 and 8 in the serum and matched synovial fluid (SF) from rheumatoid arthritis patients. The titer in the SF was lower than that in the matched plasma samples, indicating a difference in distribution of NAb to AAV depending on the body fluid compartment. This difference was more evident for AAV2, against which higher titers were measured. Of all serotypes, anti-AAV5 antibodies were the least prevalent in both the serum and SF. We next evaluated the impact of B-cell depletion on anti-AAV antibodies in rheumatoid arthritis patients who received one or two courses of the anti-CD20 antibody rituximab as part of their disease management. A drop of NAb titer was observed in a subset of those subjects carrying NAb titers ≤1:1000; however, only in a minority of subjects titers dropped below 1:5. This work provides insights into strategies to overcome the limitation of pre-existing humoral immunity to AAV vectors.

Show MeSH
Related in: MedlinePlus