Resumen de Unmasking african swine fever antigens inducing cd8+ t-cell responses with protective potential
The continuous spread of African swine fever (ASF) through Continental Europe after its introduction in Georgia in 2007, and its subsequent expansion in Asia from 2018, evidence this disease as a major threat to swine industry worldwide. ASF is a pig hemorrhagic disease of obligatory declaration to the World Organization for Animal Health (OIE) and causes enormous economic losses to the affected countries. The causative agent, African swine fever virus (ASFV), is a large, enveloped, icosahedral virus with a dsDNA genome of about 180 kbp in length. There is currently no commercial vaccine against ASFV. Early and efficient diagnosis followed by slaughtering of infected and in contact animals are the only control methods today recommended by the OIE, measures unfortunately not affordable by less favored regions.
ASF vaccine development is largely hindered by lack of knowledge about critical aspects of ASFV infection and protective immunity. In this regard, CD8+ T lymphocytes have been widely shown to play a critical role in protective response against ASFV. However, the identity of the ASFV antigens capable of inducing protective CD8+ T-cell responses remains largely unknown. Identification of such protective antigens could lead to rationale vaccine design as well as better understanding the mechanisms underlying ASFV immunity. Therefore, the present thesis aimed to determine ASFV proteins containing CD8+ T-cell epitopes with potential to elicit protective responses against the Georgia2007/1 ASFV, the isolate currently circulating in Continental Europe and Asia.
Different methodologies and strategies have been explored aiming to identify both ASFV-specific CD8+ T-cell epitopes and full-length proteins encoding promiscuously recognized CD8+ T-cell epitopes with protective potential. In order to identify ASFV-specific CD8+ T-cell epitopes, a double strategy was employed: i) a multiparametric bioinformatics analysis using the Georgia2007/1 proteome as template; and ii) an immunopeptidomic approach based on the analysis of SLA I-bound peptides found in porcine alveolar macrophages in vitro infected with ASFV. The results observed when evaluating the in vitro recognition of the peptides by ASFV-specific PBMCs obtained from ASF-recovered pigs, suggested immunopeptidomics analysis as a more reliable strategy than in silico predictions for the identification of ASFV CD8+ T-cell epitopes. As expected, peptides were not promiscuously recognized by PBMCs from all animals, confirming their marked restriction for specific SLA I alleles.
Further analysis using full-length proteins allowed determining few ASFV antigens promiscuously recognized by ASFV-specific PBMCs. Thus, stimulation of ASFV-specific PBMCs with autologous fibroblasts transiently transfected with plasmids encoding full-length ASFV ORFs fused to ubiquitin to improve SLA I antigen presentation, led to the identification of four ASFV proteins as immunodominant and promiscuously recognized antigens by ASFV-specific CD8+ T cells. Finally, DNA immunization experiments allowed demonstrating the protective potential of the ASFV antigens here identified against the Georgia2007/1 ASFV challenge.
Sterilizing protection against ASFV most likely will require a broad repertoire of B and T-cell specificities and thus, further investigations will be needed to determine other ASFV antigens eliciting protective responses. Likewise, it will most likely be indispensable the use of alternative expression platforms to encode the potential vaccine antigens, aiming to induce more solid immune response than that afforded by DNA vaccines, an ideal tool for antigen discovery but far from optimal for final veterinary vaccine formulations.
