Research paper A new antigen scanning strategy for monitoring HIV-1 specific T-cell immune responses Mauro S. Malnati a, ⁎, Silvia Heltai a , Antonio Cosma c, d , Peter Reitmeir e , Simone Allgayer c, f , Richard H. Glashoff g , Walter Liebrich g , Eftyhia Vardas h , Nesrina Imami i , Samantha Westrop i , Silvia Nozza a , Giuseppe Tambussi a , Stefano Buttò j , Emanuele Fanales-Belasio j , Barbara Ensoli j , Fabrizio Ensoli k , Antonella Tripiciano k , Claudio Fortis a , Paolo Lusso a, 1 , Guido Poli a, b , Volker Erfle c, d , Harvey Holmes l a San Raffaele Scientific Institute, Milan, Italy b University Vita e Salute, Milan, Italy c Clinical cooperation group “Immune monitoring”, Helmholtz Zentrum, München, German Research Center for Environmental Health, Neuherberg, Germany d Institute of Virology, Helmholtz Zentrum, München, German Research Center for Environmental Health, Neuherberg, Germany e Institute of Health Economics and Health Care Management, Helmholtz Zentrum, München, German Research Center for Environmental Health, Neuherberg, Germany f Institute of Virology, Technical University, Munich, Germany g Division of Medical Virology, University of Stellenbosch, South Africa h University of Witwatersrand, Faculty of Health Sciences, Johannesburg, South Africa i Imperial College-London, United Kingdom j National AIDS Center, Istituto Superiore di Sanità, Rome, Italy k S.Gallicano Hospital, IFO, Rome, Italy l Centre for AIDS Reagents (CFAR), Health Protective Agency, Potters Bar, Hertfordshire, United Kingdom article info abstract Article history: Received 30 May 2011 Received in revised form 12 September 2011 Accepted 14 September 2011 Available online xxxx Delineation of the immune correlates of protection in natural infection or after vaccination is a mandatory step for vaccine development. Although the most recent techniques allow a sensi- tive and specific detection of the cellular immune response, a consensus on the best strategy to assess their magnitude and breadth is yet to be reached. Within the AIDS Vaccine Integrated Project (AVIP http://www.avip-eu.org) we developed an antigen scanning strategy combining the empirical-based approach of overlapping peptides with a vast array of database informa- tion. This new system, termed Variable Overlapping Peptide Scanning Design (VOPSD), was used for preparing two peptide sets encompassing the candidate HIV-1 vaccine antigens Tat and Nef. Validation of the VOPSD strategy was obtained by direct comparison with 15mer or 20mer peptide sets in a trial involving six laboratories of the AVIP consortium. Cross-reactive background responses were measured in 80 HIV seronegative donors (HIV-), while sensitivity and magnitude of Tat and Nef-specific T-cell responses were assessed on 90 HIV+ individuals. In HIV-, VOPSD peptides generated background responses comparable with those of the stan- dard sets. In HIV-1+ individuals the VOPSD pools showed a higher sensitivity in detecting in- dividual responses (Tat VOPSD vs. Tat 15mers or 20mers: p ≤ 0.01) as well as in generating stronger responses (Nef VOPSD vs. Nef 20mers: p b 0.001) than standard sets, enhancing Keywords: Peptide Tat Nef ELISpot HIV-1 Vaccine Journal of Immunological Methods xxx (2011) xxx–xxx Abbreviations: VOPSD, Variable Overlapping Peptide Scanning Design;ELISpot, enzyme-linked immunospot assays;SFU, spot forming units;ICS, intracellular cytokine staining;CTL, CD8 cytotoxic T-cell. ⁎ Corresponding author at: Laboratory of Human Virology, San Raffaele Scientific Institute-DiBiT, Via Olgettina n. 58, I-20132 Milano, Italy. Tel.: +39 02 2643 4900; fax: +39 02 2643 4905. E-mail address: malnati.mauro@hsr.it (M.S. Malnati). 1 Present address: Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, United States. JIM-11364; No of Pages 11 0022-1759/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2011.09.005 Contents lists available at SciVerse ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim Please cite this article as: Malnati, M.S., et al., A new antigen scanning strategy for monitoring HIV-1 specific T-cell immune responses, J. Immunol. Methods (2011), doi:10.1016/j.jim.2011.09.005