1091. Analysis of T-Cell Responses to Helper-Dependent Serotype 5-Based Capsid-Modified Adenovirus (HD.5/35) or First-Generation Serotype 11- Based (Ad11) Vectors

Abstract

Top of pageAbstract Cellular immunity to adenovirus serotype 5 (Ad5) is an obstacle for Ad5-based gene therapy. We asked if this could be overcome by either a helper-dependent capsid-modified Ad5 vector possessing the Ad35 fiber (HD.5/35), or a first-generation vector completely derived from Ad serotype 11 (Ad11). Ad35 and Ad11 fiber containing Ad vectors efficiently transduce dendritic cells (DC) in vitro and are being evaluated for immunotherapy strategies. We transduced human peripheral blood mononuclear cells (PBMC) from 11 donors (including cancer patients) with GFP expressing Ad11 or HD.5/35 vectors, or first generation Ad5, Ad5/35 or Ad5/11 vectors as controls. Regardless of the vector used, all donors showed IFN-|[gamma]| secreting cells, detected by ELISpot assays (200|[ndash]|400 spots in 1|[times]|10e6 PBMC). The Ad-specific T-cells against the HD vector indicates a response to Ad capsid epitopes from the incoming particles. It was speculated that this could interfere with responses to a vaccination or therapy antigen expressed from Ad vectors. We evaluated this in vivo and in vitro. For in vivo studies, we injected C57Bl/6 mice transgenic for CD46 (the receptor for Ad5/35) with Ad vectors expressing the model antigen hepatitis B virus e antigen (Ad5.HBeAg, Ad5/35.HBeAg and HD.5/35.HBeAg). Fourteen days later, their splenocytes were mixed with splenocytes from na|[iuml]|ve mice in vitro pulsed with HBeAg recombinant protein, or transduced with the same or control Ad vectors. A strong anti-Ad T cell response was detected by an IFN-|[gamma]| ELISpot assay irrespective of the vector used. The frequency of HBeAg specific T cells was similar with all the vectors tested, and at least two orders of magnitude lower than that to the Ad vectors. For the in vitro human studies, we used the HD.5/35.Her2.Fc vector. It expresses a target antigen in breast cancer, Her-2/neu. We stimulated twice 5|[times]|10e7 PBMC from a normal donor with 5|[times]|10e6 transduced and matured DC. The T cell line was then stimulated with DC pulsed with a library of 168 15mer-overlapping peptides covering the extracellular domain (ECD) of Her2/neu, or transduced with a control vector (HD.5/35.GFP), or pulsed with an Ad5 hexon-derived peptide or other control peptides in an IFN-|[gamma]| ELISpot plate. A strong T cell response against Ad transduced DC and against the hexon epitope was observed. No ECD specific T cell was detected. In conclusion, i) we found a strong T cell response to Ad11 vectors, which was surprising considering previous studies from our lab and others that showed a low serum-prevalence of neutralizing anti-Ad11 antibodies in humans; this would indicate either pre-existing T-cell immunity to Ad11 or, most likely, T-cell cross-reactivity between Ad serotypes; ii) the use of HD vectors did not prevent induction of anti Ad T cells in na|[iuml]|ve animals, and the incoming Ad particles activated memory T cell responses in in vitro human studies; iii) we could not detect strong antigen specific immune responses in our experiments. Then, these studies support the idea that cellular immunity, directed to the Ad capsid epitopes, is an important problem to be solved for antigen specific vaccination/immunotherapy purposes.