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Journal of Virology, March 2008, p. 2727-2740, Vol. 82, No. 6
0022-538X/08/$08.00+0     doi:10.1128/JVI.01990-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Complement Is an Essential Component of the Immune Response to Adeno-Associated Virus Vectors

Department of Biochemistry and Molecular Biology,¹ Department of Medicine, University of Calgary, Alberta T2N 4N1, Canada,² Division of Rheumatology, University of Colorado Health Sciences Center, Denver, Colorado 80262,³ Gene Therapy Center, Columbus Children's Research Institute, Columbus Children's Hospital, Columbus, Ohio 43205⁴

Received 10 September 2007/ Accepted 2 January 2008

Adeno-associated virus (AAV) vectors are associated with relatively mild host immune responses in vivo. Although AAV induces very weak innate immune responses, neutralizing antibodies against the vector capsid and transgene still occur. To understand further the basis of the antiviral immune response to AAV vectors, studies were performed to characterize AAV interactions with macrophages. Primary mouse macrophages and human THP-1 cells transduced in vitro using an AAV serotype 2 (AAV2) vector encoding green fluorescent protein did not result in measurable transgene expression. An assessment of internalized vector genomes showed that AAV2 vector uptake was enhanced in the presence of normal but not heat-inactivated or C3-depleted mouse/human serum. Enhanced uptake in the presence of serum coincided with increased macrophage activation as determined by the expression of NF-B-dependent genes such as macrophage inflammatory protein 2 (MIP-2), interleukin-1β (IL-1β), IL-8, and MIP-1β. AAV vector serotypes 1 and 8 also activated human and mouse macrophages in a serum-dependent manner. Immunoprecipitation studies demonstrated the binding of iC3b complement protein to the AAV2 capsid in human serum. AAV2 did not activate the alternative pathway of the complement cascade and lacked cofactor activity for factor I-mediated degradation of C3b to iC3b. Instead, our results suggest that the AAV capsid also binds complement regulatory protein factor H. In vivo, complement receptor 1/2- and C3-deficient mice displayed impaired humoral immunity against AAV2 vectors, with a delay in antibody development and significantly lower neutralizing antibody titers. These results show that the complement system is an essential component of the host immune response to AAV.

Published ahead of print on 16 January 2008.

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