African swine fever virus (ASFV) is causing a worldwide pandemic affecting the porcine industry and leading to important global economic consequences. The virus causes a highly lethal hemorrhagic disease in wild boars and domestic pigs. Lack of effective vaccines hampers the control of virus spread, thus increasing the pressure on the scientific community for urgent solutions. However, knowledge on the immune components associated with protection is very limited. Here we characterized the in vitro recall response induced by immune cells from pigs intranasally vaccinated with the BA71ΔCD2 deletion mutant virus. Vaccination conferred dose-dependent cross-protection associated with both ASFV-specific antibodies and IFNγ-secreting cells. Importantly, bulk and single-cell transcriptomics of blood and lymph node cells from vaccinated pigs revealed a positive feedback from adaptive to innate immunity. Indeed, activation of Th1 and cytotoxic T cells was concomitant with a rapid IFNγ-dependent triggering of an inflammatory response characterized by TNF-producing macrophages, as well as CXCL10-expressing lymphocytes and cross-presenting dendritic cells. Altogether, this study provides a detailed phenotypic characterization of the immune cell subsets involved in cross-protection against ASFV, and highlights key functional immune mechanisms to be considered for the development of an effective ASF vaccine.

African swine fever (ASF) has become the major threat for the global swine industry. Furthermore, the epidemiological situation of African swine fever virus (ASFV) in some endemic regions of Sub-Saharan Africa is worse than ever, with multiple virus strains and genotypes currently circulating in a given area. Despite the recent advances on ASF vaccine development, there are no commercial vaccines yet, and most of the promising vaccine prototypes available today have been specifically designed to fight the genotype II strains currently circulating in Europe, Asia, and Oceania. Previous results from our laboratory have demonstrated the ability of BA71∆CD2, a recombinant LAV lacking CD2v, to confer protection against homologous (BA71) and heterologous genotype I (E75) and genotype II (Georgia2007/01) ASFV strains, both belonging to same clade (clade C). Here, we extend these results using BA71∆CD2 as a tool trying to understand ASFV cross-protection, using phylogenetically distant ASFV strains. We first observed that five out of six (83.3%) of the pigs immunized once with 10⁶ PFU of BA71∆CD2 survived the tick-bite challenge using Ornithodoros sp. soft ticks naturally infected with RSA/11/2017 strain (genotype XIX, clade D). Second, only two out of six (33.3%) survived the challenge with Ken06.Bus (genotype IX, clade A), which is phylogenetically more distant to BA71∆CD2 than the RSA/11/2017 strain. On the other hand, homologous prime-boosting with BA71∆CD2 only improved the survival rate to 50% after Ken06.Bus challenge, all suffering mild ASF-compatible clinical signs, while 100% of the pigs immunized with BA71∆CD2 and boosted with the parental BA71 virulent strain survived the lethal challenge with Ken06.Bus, without almost no clinical signs of the disease. Our results confirm that cross-protection is a multifactorial phenomenon that not only depends on sequence similarity. We believe that understanding this complex phenomenon will be useful for designing future vaccines for ASF-endemic areas.

African swine fever (ASF) is today′s number one threat for the global swine industry. Neither commercial vaccine nor treatment is available against ASF and, thus far, only live attenuated viruses (LAV) have provided robust protection against lethal ASF virus (ASFV) challenge infections. Identification of ASFV proteins inducing protective immune responses is one of the major challenges to develop safer and efficient subunit vaccines. Immunopeptidomic studies recently performed in our laboratory allowed identifying ASFV antigens recognized by ASFV-specific CD8⁺ T-cells. Here, we used data from the SLAI-peptide repertoire presented by a single set of ASFV-infected porcine alveolar macrophages to generate a complex DNA vaccine composed by 15 plasmids encoding the individual peptide-bearing ORFs. DNA vaccine priming improved the protection afforded by a suboptimal dose of the BA71ΔCD2 LAV given as booster vaccination, against Georgia2007/1 lethal challenge. Interestingly, M448R was the only protein promiscuously recognized by the induced ASFV-specific T-cells. Furthermore, priming pigs with DNA plasmids encoding M488R and MGF505-7R, a CD8⁺ T-cell antigen previously described, confirmed these two proteins as T-cell antigens with protective potential. These studies might be useful to pave the road for designing safe and more efficient vaccine formulations in the future.

The development of subunit vaccines against African swine fever (ASF) is mainly hindered by the lack of knowledge regarding the specific ASF virus (ASFV) antigens involved in protection. As a good example, the identity of ASFV-specific CD8⁺ T-cell determinants remains largely unknown, despite their protective role being established a long time ago. Aiming to identify them, we implemented the IFNγ ELISpot as readout assay, using as effector cells peripheral blood mononuclear cells (PBMCs) from pigs surviving experimental challenge with Georgia2007/1. As stimuli for the ELISpot, ASFV-specific peptides or full-length proteins identified by three complementary strategies were used. In silico prediction of specific CD8⁺ T-cell epitopes allowed identifying a 19-mer peptide from MGF100-1L, as frequently recognized by surviving pigs. Complementarily, the repertoire of SLA I-bound peptides identified in ASFV-infected porcine alveolar macrophages (PAMs), allowed the characterization of five additional SLA I-restricted ASFV-specific epitopes. Finally, in vitro stimulation studies using fibroblasts transfected with plasmids encoding full-length ASFV proteins, led to the identification of MGF505-7R, A238L and MGF100-1L as promiscuously recognized antigens. Interestingly, each one of these proteins contain individual peptides recognized by surviving pigs. Identification of the same ASFV determinants by means of such different approaches reinforce the results presented here.

African swine fever virus is the etiological agent of African swine fever, a transmissible severe hemorrhagic disease that affects pigs, causing massive economic losses. There is neither a treatment nor a vaccine available, and the only method to control its spread is by extensive culling of pigs. So far, classical vaccine development approaches have not yielded sufficiently good results in terms of concomitant safety and efficacy. Nowadays, thanks to advances in genomic and proteomic techniques, a reverse vaccinology strategy can be explored to design alternative vaccine formulations. In this study, ASFV protein sequences were analyzed using an in-house pipeline based on publicly available immunoinformatic tools to identify epitopes of interest for a prospective vaccine ensemble. These included experimentally validated sequences from the Immune Epitope Database, as well as de novo predicted sequences. Experimentally validated and predicted epitopes were prioritized following a series of criteria that included evolutionary conservation, presence in the virulent and currently circulating variant Georgia 2007/1, and lack of identity to either the pig proteome or putative proteins from pig gut microbiota. Following this strategy, 29 B-cell, 14 CD4⁺ T-cell and 6 CD8⁺ T-cell epitopes were selected, which represent a starting point to investigating the protective capacity of ASFV epitope-based vaccines.

African swine fever (ASF) has become the major threat to the global swine industry. Lack of available commercial vaccines complicates the implementation of global control strategies. So far, only live attenuated ASF viruses (ASFV) have demonstrated solid protection efficacy at the experimental level. The implementation of molecular techniques has allowed the generation of a collection of deletion mutants lacking ASFV-specific virulence factors, some of them with promising potential as vaccine candidates against the pandemic genotype II ASFV strain currently circulating in Africa, Europe, Asia and Oceania. Despite promising results, there is room for improvement, mainly from the biosafety point of view. Aiming to improve the safety of BA71∆CD2, a cross-protective recombinant live attenuated virus (LAV) lacking the ASFV CD2v gene (encoding β-glucuronidase as a reporter gene) available in our laboratory, three new recombinants were generated using BA71∆CD2 as a template: the single mutant BA71∆CD2f, this time containing the fluorescent mCherry reporter gene instead of CD2v, and two double recombinants lacking CD2v and either the lectin gene (EP153R) or the uridine kinase (UK) gene (DP96R). Comparative in vivo experiments using BA71∆CD2f, BA71∆CD2DP96R and BA71∆CD2EP153R recombinant viruses as immunogens, demonstrated that deletion of either DP96R or EP153R from BA71∆CD2f decreases vaccine efficacy and does not improve safety. Our results additionally confirm ASFV challenge as the only available method today to evaluate the protective efficacy of any experimental vaccine. We believe that understanding the fine equilibrium between attenuation and inducing protection in vivo deserves further study and might contribute to more rational vaccine designs in the future.