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Additional file 1. Instructions to the patient for intimate hygiene.

Flavia Ferrantelli,1,* Claudia Arenaccio,1,* Francesco Manfredi,1 Eleonora Olivetta,1 Chiara Chiozzini,1 Patrizia Leone,1 Zulema Percario,2 Alessandro Ascione,1 Michela Flego,1 Paola Di Bonito,3 Luisa Accardi,3 Maurizio Federico1 1National Center for Global Health, Istituto Superiore Di Sanità (ISS), Rome, Italy; 2Department of Science, Roma Tre University, Rome, Italy; 3Department of Infectious Diseases, Istituto Superiore Di Sanità (ISS), Rome, Italy*These authors contributed equally to this workCorrespondence: Maurizio FedericoNational Center for Global Health, Istituto Superiore Di Sanità, Viale Regina Elena, 299, Rome 00161, ItalyTel +39-06-4990-3605Fax +39-06-49903210Email maurizio.federico@iss.itPurpose: Single-chain variable fragments (scFvs) are one of the smallest antigen-binding units having the invaluable advantage to be expressed by a unique short open reading frame (ORF). Despite their reduced size, spontaneous cell entry of scFvs remains inefficient, hence precluding the possibility to target intracellular antigens. Here, we describe an original strategy to deliver scFvs inside target cells through engineered extracellular vesicles (EVs). This approach relies on the properties of a Human Immunodeficiency Virus (HIV)-1 Nef mutant protein referred to as Nefmut. It is a previously characterized Nef allele lacking basically all functions of wt Nef, yet strongly accumulating in the EV lumen also when fused at its C-terminus with a foreign protein. To gain the proof-of-principle for the efficacy of the proposed strategy, the tumor-promoting Human Papilloma Virus (HPV)16-E7 protein was considered as a scFv-specific intracellular target. The oncogenic effect of HPV16-E7 relies on its binding to the tumor suppressor pRb protein leading to a dysregulated cell duplication. Interfering with this interaction means impairing the HPV16-E7-induced cell proliferation.Methods: The Nefmut gene was fused in frame at its 3ʹ-terminus with the ORF coding for a previously characterized anti-HPV16-E7 scFv. Interaction between the Nefmut-fused anti-HPV16-E7 scFv and the HPV16-E7 protein was tested by both confocal microscope and co-immunoprecipitation analyses on co-transfected cells. The in cis anti-proliferative effect of the Nefmut/anti-HPV16-E7 scFv was assayed by transfecting HPV16-infected cells. The anti-proliferative effect of EVs engineered with Nefmut/anti-HPV16-E7 scFv on HPV16-E7-expressing cells was evaluated in two ways: i) through challenge with purified EVs by a Real-Time Cell Analysis system and ii) in transwell co-cultures by an MTS-based assay.Results: The Nefmut/anti-HPV16-E7 scFv chimeric product is efficiently uploaded in EVs, binds HPV16-E7, and inhibits the proliferation of HPV16-E7-expressing cells. Most important, challenge with cell-free EVs incorporating the Nefmut/anti-HPV16-E7 scFv led to the inhibition of proliferation of HPV16-E7-expressing cells. The proliferation of these cells was hindered also when they were co-cultured in transwells with cells producing EVs uploading Nefmut/anti-HPV16-E7 scFv.Conclusion: Our data represent the proof-of-concept for the possibility to target intracellular antigens through EV-mediated delivery of scFvs. This finding could be relevant to design novel methods of intracellular therapeutic interventions.Keywords: single-chain variable fragments, Human Papilloma Virus E7 protein, exosomes, human immunodeficiency virus Nef protein, scFv intracellular delivery

// Carla Amici 1 , Michela Visintin 2 , Francesca Verachi 1,3 , Francesca Paolini 4 , Zulema Percario 5 , Paola Di Bonito 3 , Angela Mandarino 3 , Elisabetta Affabris 5 , Aldo Venuti 4 and Luisa Accardi 3 1 University of Rome Tor Vergata, Department of Biology, Via della Ricerca Scientifica, Rome, Italy 2 Rottapharm Biotech Srl, Biotherapeutics Discovery, Area Science Park – Basovizza, Trieste, Italy 3 Istituto Superiore di Sanita, Department of Infectious, Parasitic and Immunomediated Diseases, Viale Regina Elena, Rome, Italy 4 Regina Elena National Cancer Institute, Laboratory of Virology and HPV-Unit, via delle Messi d’Oro, Rome, Italy 5 University Roma Tre, Department of Science, Viale G. Marconi, Rome, Italy Correspondence to: Luisa Accardi, email: // Keywords : E6 oncoprotein, human papillomaviruses, cancer therapy, scFv, intracellular antibodies Received : September 03, 2015 Accepted : January 04, 2016 Published : January 15, 2016 Abstract Single-chain variable fragments (scFvs) expressed as “intracellular antibodies” (intrabodies) can target intracellular antigens to hamper their function efficaciously and specifically. Here we use an intrabody targeting the E6 oncoprotein of Human papillomavirus 16 (HPV16) to address the issue of a non-invasive therapy for HPV cancer patients. A scFv against the HPV16 E6 was selected by Intracellular Antibody Capture Technology and expressed as I7nuc in the nucleus of HPV16-positive SiHa, HPV-negative C33A and 293T cells. Colocalization of I7nuc and recombinant E6 was observed in different cell compartments, obtaining evidence of E6 delocalization ascribable to I7nuc. In SiHa cells, I7nuc expressed by pLNCX retroviral vector was able to partially inhibit degradation of the main E6 target p53, and induced p53 accumulation in nucleus. When analyzing in vitro activity on cell proliferation and survival, I7nuc was able to decrease growth inducing late apoptosis and necrosis of SiHa cells. Finally, I7nuc antitumor activity was demonstrated in two pre-clinical models of HPV tumors. C57BL/6 mice were injected subcutaneously with HPV16-positive TC-1 or C3 tumor cells, infected with pLNCX retroviral vector expressing or non-expressing I7nuc. All the mice injected with I7nuc-expressing cells showed a clear delay in tumor onset; 60% and 40% of mice receiving TC-1 and C3 cells, respectively, remained tumor-free for 17 weeks of follow-up, whereas 100% of the controls were tumor-bearing 20 days post-inoculum. Our data support the therapeutic potential of E6-targeted I7nuc against HPV tumors.