Journal of Medical Virology 81:211–216 (2009) In Vitro Analysis of Synergism and Antagonism of Different Nucleoside/Nucleotide Analogue Combinations on the Inhibition of Human Immunodeficiency Virus Type 1 Replication M. Perez-Olmeda, 1 J. Garcia-Perez, 1 E. Mateos, 1 S. Spijkers, 1 M.C. Ayerbe, 2 A. Carcas, 3 and J. Alcami 1 * 1 Unidad de Inmunopatologı´a del SIDA,Centro Nacional de Microbiologı´a, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain 2 Centro de Salud Adelfas, A ´ rea Sanitaria 1, Servicio Madrilen ˜ o de Salud, Madrid, Spain 3 Centro de Farmacologı´a Clı´nica, Hospital Universitario La Paz, Facultad de Medicina, UAM, Madrid, Spain In this study we have developed an in vitro system to evaluate the combined effect of two NRTIs on HIV replication and to assess their antagonism or synergy. Synergy or antagonism effect was determined in peripheral blood mononuclear cells (PBMCs) to approach a more physiological model than T-cell lines. PBMCs were infected with a full-length HIV-1 clone carrying the luciferase gene as a reporter. The following combinations were investigated: zidovudineþstavudine (ZDV þ d4T), lamivudine þ abacavir (3TC þ ABC), lamivu- dine þ didanosine (3TC þ ddI), lamivudine þ stavudine (3TC þ d4T), tenofovir þ stavudine (TDF þ d4T), tenofovir þ didanosine (TDF þ ddI), tenofovir þ abacavir (TDF þ ABC), tenofovir þ lamivudine (TDF þ 3TC), tenofovir þ zidovudine (TDF þ ZDV), stavudine þ didanosine (d4T þ ddI), zidovudine þ lamivudine (ZDV þ 3TC), abacavir þ didanosine (ABC þ ddI), zidovudine þ didanosine (ZDV þ ddI), and abacavir þ stavudine (ABC þ d4T). The effect of combining two drugs was evaluated with a quantitative method based on the median-effect principle of Chou and Talalay. A synergistic effect was observed with combina- tions containing TDF and ZDV or d4T, d4T and ddI and ZDV plus 3TC. In contrast, combinations including TDF þ ddI, 3TC þ ddI, ABC þ ddI, and ZDV þ ddI showed an antagonistic effect on the inhibition of viral replication at all levels of inhibition tested. Lower antagonistic effect was also found in drug combinations that included 3TC þ ABC, 3TC þ TDF, 3TC þ d4T, and TDF þ ABC. In conclusion, the method developed allows to measure in vitro the effect of different combi- nations of two NRTIs on HIV replication. The results suggest that combined therapy including TDF with thymidine analogues may be consid- ered for future therapeutic options in contrast to clearly antagonistic combinations such us TDF plus ddI or 3TC plus ddI, that would explain virological failure in clinical studies when these combinations were used. J. Med. Virol. 81:211–216, 2009. ß 2008 Wiley-Liss, Inc. KEY WORDS: HIV; antiretroviral therapy; syn- ergism; antagonism INTRODUCTION Currently, treatment with two nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) plus a non- NRTI (NNRTI) or a protease inhibitor (PI) represents effective and safe combination therapy for human immunodeficiency virus type-1 (HIV-1) infection [Ray et al., 2005; Hammer et al., 2006]. The approval of a large number of NRTIs for the treatment of HIV-1 infection has permitted the use of new combinations that have not been evaluated previously in clinical trials. The goal of these regimens is to facilitate its administration in order to improve adherence of patients to treatment and to decrease side effects while maintaining full efficacy and M. Perez-Olmeda and J. Garcia-Perez contributed equally to this work. Grant sponsor: Red de Investigacio ´ n en SIDA (partial support); Grant number: RIS ISCIII-RETIC RD06/006; Grant sponsor: Fundacio ´n para la investigacio ´n y prevencio ´n del SIDA en Espan ˜ a; Grant number: FIPSE 3074/99; Grant sponsor: Fondo de Investigacion Sanitaria; Grant number: FIS PI040525; Grant sponsor: Instituto de Salud Carlos III, Ministerio de Sanidad (ISCIII INTRAMURAL); Grant number: ISCIII 03/ESP27; Grant sponsor: Juan de la Cierva Programme of Ministerio de Educacio ´n y Ciencia (to M.P.-O.); Grant sponsor: Instituto de Salud Carlos III (to J.G.-P.); Grant number: 00/0034. *Correspondence to: J. Alcami, AIDS Immunopathology Unit, National Center of Microbiology, Instituto de Salud Carlos III. Ctra. Majadahonda-Pozuelo, km 2, 28220 Majadahonda, Madrid, Spain. E-mail: ppalcami@isciii.es Accepted 22 September 2008 DOI 10.1002/jmv.21377 Published online in Wiley InterScience (www.interscience.wiley.com) ß 2008 WILEY-LISS, INC.