THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE J Gene Med 2005; 7: 432–441. Published online 28 October 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.678 Immune responses following salivary gland administration of recombinant adeno-associated virus serotype 2 vectors Marc R. Kok 1,2 * Antonis Voutetakis 1 Seiichi Yamano 1 Jianghua Wang 1 Ana Cotrim 1 Hisako Katano 1 Ioannis Bossis 1 John A. Chiorini 1 Simon D. Tran 1 Paul P. Tak 2 Bruce J. Baum 1 1 Gene Therapy and Therapeutics Branch, NIDCR, NIH, DHHS, Bethesda, MD, USA 2 Division of Clinical Immunology and Rheumatology, AMC, University of Amsterdam, Amsterdam, The Netherlands *Correspondence to: Marc R. Kok, Academic Medical Center, Division of Clinical Immunology and Rheumatology, Meibergdreef 9, Room F4-218, 1105 AZ Amsterdam, The Netherlands. E-mail: m.r.kok@amc.uva.nl Received: 22 January 2004 Revised: 13 August 2004 Accepted: 24 August 2004 Abstract Background Gene transfer to salivary glands (SGs) can be accomplished in a minimally invasive manner, resulting in stable, long-term secretion of the transgene product. Therefore, SGs provide a novel target site for several potentially useful clinical gene therapeutics applications. Previous studies have indicated that intravenous, intramuscular and intranasal administration of recombinant adeno-associated virus serotype 2 (rAAV2) vectors induce host immune responses. There are no reported studies on immune responsiveness of rAAV2 vector administration to SGs. Material and methods Vectors were administered by retrograde infusion to the SGs of Balb/c mice in various combinations. Thereafter, transgene expression was determined, and evaluations of host innate and adaptive immune responsiveness performed over a 56-day period. Results Histological examination of SGs from vector-treated mice showed no significant changes in appearance from controls, including the frequency of activated macrophage detection. There were also no differences in salivary flow rates among experimental groups. In vitro stimulation of splenocytes from mice administered rAAV2 showed elevated interferon-γ levels in culture media. Significant titers of neutralizing antibodies to rAAV2 were detected in serum of mice following rAAV2 vector administration. While SGs could be transduced with low doses of vector it was not possible to repeat the administration and detect transduction with the same serotype at low doses. However, repeat administration was possible with an alternative serotype (rAAV4). Conclusions Following a single administration of rAAV2 vectors to SGs there is no significant innate immune response. However, rAAV2 vector administration to SGs results in both cellular and humoral immune responses. The latter may interfere with the efficacy of repeated rAAV2 vector administration. Copyright  2004 John Wiley & Sons, Ltd. Keywords rAAV2; immune response; gene transfer; salivary glands; readministration Introduction Salivary glands (SGs) provide a novel target site for several potentially useful clinical gene transfer applications [1]. SGs are capable of producing large amounts of proteins, and are a site where gene transfer can be readily Copyright  2004 John Wiley & Sons, Ltd.