Successful attenuation of humoral immunity to viral capsid and transgenic protein following AAV-mediated gene transfer with a non-depleting CD4 antibody and cyclosporine - PubMed (original) (raw)

Successful attenuation of humoral immunity to viral capsid and transgenic protein following AAV-mediated gene transfer with a non-depleting CD4 antibody and cyclosporine

J H McIntosh et al. Gene Ther. 2012 Jan.

Abstract

The ability of transient immunosuppression with a combination of a non-depleting anti-CD4 (NDCD4) antibody and cyclosporine (CyA) to abrogate immune reactivity to both adeno-associated viral vector (AAV) and its transgene product was evaluated. This combination of immunosuppressants resulted in a 20-fold reduction in the resulting anti-AAV8 antibody titres, to levels in naïve mice, following intravenous administration of 2 × 10(12) AAV8 vector particles per kg to immunocompetent mice. This allowed efficient transduction upon secondary challenge with vector pseudotyped with the same capsid. Persistent tolerance did not result, however, as an anti-AAV8 antibody response was elicited upon rechallenge with AAV8 without immunosuppression. The route of vector administration, vector dose, AAV serotype or the concomitant administration of adenoviral vector appeared to have little impact on the ability of the NDCD4 antibody and CyA combination to moderate the primary humoral response to AAV capsid proteins. The combination of NDCD4 and CyA also abrogated the humoral response to the transgene product, that otherwise invariably would occur, following intramuscular injection of AAV5, leading to stable transgene expression. These observations could significantly improve the prospects of using rAAV vectors for chronic disorders by allowing for repeated vector administration and avoiding the development of antibodies to the transgene product.

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Conflict of interest statement

Conflicts/Disclosures

Hermann Waldmann and Stephen Cobbold are shareholders in TolerRx Inc. The other authors have no financial conflicts of interest to declare.

Figures

Figure 1. Successful attenuation of humoral response following transient immunosuppression

(A) Schema of immunosuppression. C57Bl/6 mice (N=7–8/group) received immunosuppression on days −1, 0, 1, 3, 6, 8 and 2×1012/kg AAV8 LP1 hFIX on day 0. (B) Normal levels of CD4+ T cells in the peripheral blood of mice CyA/NDC4 when compared to untreated animals (n=2 in each group) as shown by specific staining for CD3 (PE) and CD4 (FITC). (C) Transduction efficiency is unaffected by concurrent administration of immunosuppression. hFIX levels were measured by ELISA on day 98 (n=6) mean ±SEM (D) Attenuation of humoral response to capsid by immunosuppression. Anti-AAV antibody was measure by ELISA on day 98 (n=6). Results shown as mean ±SEM.

Figure 1. Successful attenuation of humoral response following transient immunosuppression

Figure 2. Re-administration of AAV8

(A) Successful transduction with the same serotype of AAV. 2×1012/kg AAV8 CAGG IFNβ was given on day 100, 20 days later IFNβ levels were measured by ELISA (n=7, mean ±SEM). (B) Semi-quantitative PCR confirming selective transduction. 250ng of liver DNA from (n=2) CyA+Ab, NT (no immunosuppression), Naïve and IFN (hIFNβ only) cohorts were analysed with primers against hFIX, hIFNβ and βactin (C) Mice are not tolerised to capsid protein following transient immunosuppression. Anti-AAV8 abs were determined by ELISA on day 60 following 2nd administration n=6 mean ±SEM. (D) Mice were challenged with AAV8 LP1 hFX 5×1011/kg on day 296, 21 days later mice were bleed and FX levels were determined by ELISA n=3

Figure 2. Re-administration of AAV8

Figure 3. Attenuation of immune response after wild type AAV infection

Balb/c mice were treated with 4×1012/kg wt-AAV2 alone or with 4×1012/kg Ad GFP. 4 weeks latter anti-AAV2 Abs were measured by ELISA n=3 mean ±SEM.

Figure 4. Prevention of humoral response to transgene and capsid following intra muscular administration

(A) Attenuation of humoral immunity to transgene after intra muscular delivery of 4×1011vg/kg rAAV2/5 CAGG hFIX. Transgene levels measured by ELISA at day 21 n=3 mean ±SEM. (B) Attenuation of humoral response to capsid after intra muscular administration of vector. Anti-AAV5 antibodies detected by ELISA at day 21 n=3 mean ±SEM. (C) Anti –hFIX antibody response is prevented with the use of transient immunosupression. hFIX antibody was measured by ELISA on day 21 n=3 mean ±SEM.

Figure 4. Prevention of humoral response to transgene and capsid following intra muscular administration

Figure 5. AAV capsid or hFIX specific T cells at 6 months after intramuscular delivery of rAAV2/5 CAGG hFIX

A. Capsid (AAV5) or hFIX specific IFN-γ ELISpot; results are expressed in spot forming units (SFU)/106 splenocytes as average (± SD) of 3 replicates. Media = negative control; and PMA, positive control. B A representative ELISpot assay showing difference in the number of IFN-γ expressing AAV5 capsid specific T cells in splenocytes obtained from AAV untreated (-ve control) or AAV5 transduced animals with (AAV5+CyA+Ab) or without immunosuppression (AAV5 alone).

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Successful attenuation of humoral immunity to viral capsid and transgenic protein following AAV-mediated gene transfer with a non-depleting CD4 antibody and cyclosporine - PubMed (original) (raw)