A novel dual promoter DNA vaccine induces CD8 + response against Toxoplasma gondii sporozoite specific surface protein ‘‘SporoSAG’’ through non-apoptotic cells Sultan Gülçe _ Iz a,b,1 , Mert Dös ßkaya b,⇑,1 , Ays ße Caner b , Aysu Deg ˘irmenci Dös ßkaya b , Fernando Rodriguez c , Yüksel Gürüz b , S. _ Ismet Delilog ˘lu Gürhan a a Ege University, Faculty of Engineering, Department of Bioengineering, Bornova, Izmir 35100, Turkey b Ege University Medical School, Department of Parasitology, Center for Vaccine Development, Bornova, Izmir 35100, Turkey c Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autonoma de Barcelona, Bellaterra, 08193 Barcelona, Spain article info Article history: Received 29 December 2013 Revised 9 April 2014 Accepted 16 April 2014 Keywords: Toxoplasma gondii DNA vaccine SporoSAG Bcl-xL anti-apoptotic protein abstract Toxoplasma gondii is a protozoan parasite that can infect all warm blooded animals including humans. During natural course of T. gondii infection, bradyzoites or sporozoites invade the intestinal cells and turn into tachyzoite form in 12–18 h. Therefore, a vaccine against toxoplasmosis is required to induce protec- tive immune response initially in intestines against bradyzoites or sporozoites. The present study aimed to generate a DNA vaccine containing a sporozoite specific surface antigen ‘‘SporoSAG’’. To increase anti- gen specific-CD8 + T lymphocyte response, anti-apoptotic Bcl-xL gene was inserted to vaccine as a molec- ular adjuvant. For the construction of DNA vaccine, SporoSAG gene was inserted after CMV promoter and Bcl-xL gene was inserted in frame with EGFP after IRES promoter (pSporoSAG/Bcl-xL). Bcl-xL expression and func- tionality as well as humoral and cellular immune responses were demonstrated by Western blotting and flow cytometer. Western blotting and flow cytometer analyses rationalized Bcl-xL expression that impedes apoptotic cell death. The ratio of pSporoSAG/Bcl-xL transfected cells were significantly higher than empty pEGFP control plasmid (P < 0.05). Analysis of sera obtained from vaccinated mice showed strong anti-SporoSAG specific IgG response. The ratio of CD8 + T lymphocytes secreting IFN-c significantly increased compared to controls (P < 0.0001), indicative of protection against toxoplasmosis. The results of this study reveal the ability of SporoSAG protein to induce CD8 + T lymphocyte response for the first time. Overall, SporoSAG protein can be included to multivalant vaccine formulations in future studies to increase the protection in infections acquired through T. gondii oocysts. Ó 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Introduction Toxoplasma gondii is an obligate intracellular parasite infecting all warm-blooded animals, including humans. T. gondii infection causes congenital toxoplasmosis in newborns that may lead to fetal anomalies, retinochoroiditis leading to blindness, lethal toxo- plasmic encephalitis in immune compromised patients, and organ failure in transplantation patients. Currently, toxoplasmosis is linked to schizophrenia and other mental disorders [1,2]. Worldwide, 500 million people are estimated to be infected with T. gondii. Infection rates among pregnant women range from 37% to 58% in Europe, 10.8% in USA, and 30 to 60% in Turkey [3–6]. In United States, 400–4000 infants are estimated to born with con- genital toxoplasmosis each year and up to 1,26 million cases of ocular toxoplasmosis are detected [6,7]. The present drugs are not 100% effective against toxoplasmosis and thus, a vaccine appears as an alternative in protection against T. gondii infections. Finally, after classification of T. gondii oocysts in category B bioterrorism agents as a water safety threat, the demand for a protective vaccine against toxoplasmosis has increased [8]. This remark was supported by tainted water out- breaks such as the British Columbia, Great Victoria outbreak (affected 7718 people), Santa Isabel do Ivai outbreak (located in http://dx.doi.org/10.1016/j.trivac.2014.04.003 1879-4378/Ó 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). ⇑ Corresponding author. Tel.: +90 232 390 4734; fax: +90 232 388 1347. E-mail address: mert.doskaya@ege.edu.tr (M. Dös ßkaya). 1 These authors contributed equally to this work. Trials in Vaccinology 3 (2014) 81–88 Contents lists available at ScienceDirect Trials in Vaccinology journal homepage: www.elsevier.com/locate/trivac