Charles Vragniau, Jens-Martin Hübner, Peter Beidler, Sucheol Gil, Kamola Saydaminova, Zhuo-Zhuang Lu, Roma Yumul, Hongjie Wang, Maximilian Richter, Pavel Sova, Charles Drescher, Pascal Fender, André Lieber

Defensins are small anti-microbial peptides capable of neutralizing human adenovirus (HAdV) in vitro by binding capsid proteins and blocking endosomal escape of virus. In humans, the alpha defensin HD5 is produced by specialized epithelial cells of the gastro-intestinal and genito-urinary tracts. Here we demonstrate that HD5 is also expressed as an active, secreted peptide by epithelial ovarian and lung cancer cells in situ, in patient biopsies. This finding triggered us to study the role of HD5 in infection and spread of replication-competent, oncolytic HAdV type 3 virus. HAdV-3 produces large amounts of penton-dodecahedra (PtDd), virus-like particles during replication. We have previously shown that PtDd are involved in opening epithelial junctions thus facilitating lateral spread of de novo produced virions. Here we describe a second function of PtDd, namely the blocking of HD5. A central tool to prove that viral PtDd neutralize HD5 and support spread of progeny virus was a HAdV-3 mutant virus that was disabled for forming PtDd (mut-Ad3GFP). We demonstrated that viral spread of mut-Ad3GFP was blocked by synthetic HD5 whereas that of the wild-type form (wt-Ad3GFP) was only minimally impacted. In human colon cancer Caco-2 cells, induction of cellular HD5 expression by fibroblast growth factor 9 (FGF9) significantly inhibited viral spread and progeny virus production for mut-Ad3GFP but not for wt-Ad3GFP. Finally, the ectopic expression of HD5 in tumor cells diminished the in vivo oncolytic activity of mut-Ad3GFP but not wt-Ad3GFP. These data suggest a new mechanism of HAdV-3 to overcome innate antiviral host responses. Our study has implications for oncolytic adenovirus therapy. Previously, it has been reported that human defensin HD5 inactivates specific human adenoviruses by binding to capsid proteins and blocking endosomal escape of virus. The central new findings described in our manuscript are: i) the discovery of a new mechanism that human adenovirus serotype 3 uses to overcome innate antiviral host responses mediate by human defensin HD5. This mechanism is based on the capacity of Ad3 to produce subviral penton-dodecahedral particles that act as decoys for HD5 and thus prevent the inactivation of virus progeny produced upon replication, ii) the demonstration that ectopic HD5 expression in cancer cells decreases the oncolytic efficacy of a serotype 5 based adenovirus vector and iii) the demonstration that epithelial ovarian and lung cancers express HD5. The study improves our understanding on how adenoviruses establish infection in epithelial tissues and has implications for cancer therapy with oncolytic adenoviruses.