In vivo cross-protection seemed to correlate with the ability of immunized pigs to induce specific CD8+ T-cells capable to recognize homologous and heterologous ASFV strains [12]. antigens. Interestingly, each one of these proteins contain individual peptides identified by surviving pigs. Recognition of the same ASFV determinants by means of such different methods reinforce the results offered here. genus in an asymptomatic manner [3]. Since then, ASF remained endemic in many sub-Saharan countries with sporadic exportations to additional continents. Two ASFV entries in Portugal, dated in 1957 and 1960, provoked 40 years of ASFV endemicity in the Iberian peninsula, the sporadic event of ASF in some countries of Europe and South America and the establishment of ASFV in Sardinia since 1978 until today [4]. Continental Europe became free of ASF in 1997, but only 10 years later on, in 2007, ASFV reentered Europe through Georgia, rapidly expanding to neighboring countries of Eastern Europe [5]. In 2014, the computer virus entered the European Union (EU) territory for the Ivacaftor hydrate first time, influencing both home pigs and crazy boars, the second option playing a critical part in ASF spread. In this area, the main causes of ASFV transmission include pig to pig contact, infected pig products, or infected fomites, such as transport vehicles [4]. Conversely, crazy boar-mediated transmission has been considered a minor risk factor in Asia, albeit this look at is currently becoming revised, with some countries reporting relevant outbreaks in their crazy boar populations [6]. Since its 1st declaration in China in 2018, most probably due to the importation of contaminated pork products, ASFV offers expanded extremely fast to all neighboring countries, reaching more than 28 countries from Asia and Oceania, causing an economic crisis of gigantic proportions [7,8]. Consequently, Ivacaftor hydrate developing safe and efficacious vaccines against ASF is definitely a priority for the swine market worldwide [9]. Immunization with recombinant live attenuated viruses (LAV) conferred safety against experimental challenge with genotype Ivacaftor hydrate II ASFV strains, currently circulating in Europe and Asia [10,11,12,13,14]. Regrettably, the molecular and immunological mechanisms eliciting this immunity are poorly recognized, albeit innate immune reactions [15,16,17,18], and both ASFV-specific antibodies [19,20] and CD8+ T-cells [21], may play complementary functions. CD8+ T-cell reactions, in the absence of antibodies, have demonstrated to be responsible for the partial safety induced by DNA vaccines in the absence of antibodies [22,23]. However, the safety afforded so far has been limited to homologous lethal challenge with E75 (genotype I) [22,23], and offers proved unsuccessful against experimental challenge with Georgia2007/1 [24]. In addition, the difficulty of ASFV, encoding more than CFD1 150 proteins [25,26,27,28], difficulties the recognition of the specific antigens and determinants inducing protecting reactions. The aim of this study was to explore the effectiveness of three different strategies to identify ASFV CD8+ T-cell epitopes and ASFV proteins, offered in the SLA I-context and promiscuously identified by CD8+ T-cells from ASF survivors. The detection of ASFV-specific T-cells was assessed by IFN ELISpot, using peripheral blood mononuclear cells (PBMCs) as effector cells from pigs experimentally vaccinated with BA71CD2 [12] and surviving the infection with Geogia2007/1, the virulent ASFV globally circulating. Different stimuli were utilized for the ELISpot assay: (i) synthetic peptides selected by in silico predictions; (ii) synthetic peptides selected by immunopeptidomics; or (iii) autologous fibroblasts transfected with plasmids encoding individual full-length open reading frames (ORFs) fused to ubiquitin [22,23,29,30]. Together with a complete list of ASFV peptides susceptible to become offered in the SLA I context, here, we statement a collection of specific peptides and proteins that are specifically identified by T-cells from ASF surviving pigs. Furthermore, the three ASFV antigens characterized as promiscuously inducing specific T-cell reactions (independently of the SLA I haplotype), were concomitantly recognized by the different methods here implemented. 2. Materials and Methods 2.1. Cells and Viruses Porcine alveolar macrophages (PAMs) from healthy standard pigs (Landrace Large White) were acquired by bronchoalveolar lung lavage. Porcine PBMCs were isolated from.