Article M448R and MGF505-7R: Two African Swine Fever Virus Antigens Commonly Recognized by ASFV-Specific T-Cells and with Protective Potential Laia Bosch-Camós 1,2 , Elisabet López 1,2 , Javier Collado 3 , María J. Navas 1,2 , Miguel Blanco-Fuertes 1,2 , Sonia Pina-Pedrero 1,2 , Francesc Accensi 1,4 , Maria Luisa Salas 5 , Egbert Mundt 6 , Veljko Nikolin 6 and Fernando Rodríguez 1,2, * 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; laia.bosch@irta.cat (L.B.-C.); elisabeth.lopezf@gmail.com (E.L.); mariajesus.navas@irta.cat (M.J.N.); miguel.blanco@irta.cat (M.B.-F.); sonia.pina@irta.cat (S.P.-P.); francesc.accensi@irta.cat (F.A.) 2 OIE Collaborating Centre for the Research and Control of Emerging and Re-Emerging Swine Diseases in Europe (IRTA-CReSA), 08193 Bellaterra, Spain 3 Departament de Biologia Cellular, Fisiologia i Immunologia, Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; javiercolladomiguens@gmail.com 4 UAB, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 5 Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad   Autònoma de Madrid, 28049 Madrid, Spain; mlsalas@cbm.csic.es 6 Boehringer Ingelheim Veterinary Research Center (BIVRC) GmbH & Co. KG, 30559 Hannover, Germany; Citation: Bosch-Camós, L.; López, E.; egbert.mundt@boehringer-ingelheim.com (E.M.); veljko.nikolin@boehringer-ingelheim.com (V.N.) Collado, J.; Navas, M.J.; * Correspondence: fernando.rodriguez@irta.cat Blanco-Fuertes, M.; Pina-Pedrero, S.; Accensi, F.; Salas, M.L.; Mundt, E.; Abstract: African swine fever (ASF) is today0 s number one threat for the global swine industry. Nikolin, V.; et al. M448R and Neither commercial vaccine nor treatment is available against ASF and, thus far, only live attenuated MGF505-7R: Two African Swine viruses (LAV) have provided robust protection against lethal ASF virus (ASFV) challenge infections. Fever Virus Antigens Commonly Identification of ASFV proteins inducing protective immune responses is one of the major challenges Recognized by ASFV-Specific T-Cells and with Protective Potential. to develop safer and efficient subunit vaccines. Immunopeptidomic studies recently performed in Vaccines 2021, 9, 508. https:// our laboratory allowed identifying ASFV antigens recognized by ASFV-specific CD8+ T-cells. Here, doi.org/10.3390/vaccines9050508 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 Academic Editor: Romain Paillot individual peptide-bearing ORFs. DNA vaccine priming improved the protection afforded by a suboptimal dose of the BA71∆CD2 LAV given as booster vaccination, against Georgia2007/1 lethal Received: 29 March 2021 challenge. Interestingly, M448R was the only protein promiscuously recognized by the induced Accepted: 12 May 2021 ASFV-specific T-cells. Furthermore, priming pigs with DNA plasmids encoding M488R and MGF505- Published: 14 May 2021 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 Publisher’s Note: MDPI stays neutral efficient vaccine formulations in the future. with regard to jurisdictional claims in published maps and institutional affil- Keywords: African swine fever; antigen discovery; T-cells; DNA immunization; live attenuated iations. virus; protection; immunopeptidomics Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. This article is an open access article African swine fever (ASF), a pig hemorrhagic disease of obligatory notification to distributed under the terms and the World Organization for Animal Health (OIE), is currently one of the major threats to conditions of the Creative Commons domestic pigs worldwide. While ASF has been enzootic in many Sub-Saharan countries Attribution (CC BY) license (https:// since its discovery in 1921, the last occurrence of ASF virus (ASFV) in Georgia in 2007 creativecommons.org/licenses/by/ has provoked its global expansion, starting first in the Caucasian region and Russia and 4.0/). reaching countries of the European Union in 2014 [1,2]. ASFV in this area has circulated Vaccines 2021, 9, 508. https://doi.org/10.3390/vaccines9050508 https://www.mdpi.com/journal/vaccines Vaccines 2021, 9, 508 2 of 16 between domestic pigs and wild boars, equally susceptible to ASFV, complicating the control of the disease [3]. The expansion of ASF in Asia and Oceania has been much faster than in Europe since it was first detected in China in Summer 2018 [4,5], mostly mediated by human trading activities, with wild boars playing a marginal role thus far. Due to the lack of commercial vaccines and therapeutics, classic sanitary methods remain as the only control strategy for ASF. These include early diagnosis followed by stamping out of the infected and potentially in contact animals, extensive disinfection and rigorous biosecurity protocols on farms [6]. Confirmation of ASF represents major trade restrictions and serious economic losses for the affected regions [7,8]. Therefore, developing safe and efficient vaccines against ASF is imperative if aiming to control and eradicate the disease. The complexity of ASFV, a large, multi-enveloped and multi-capsid, icosahedral virus with a dsDNA genome of about 180 kbp in length [9], has complicated the generation of safe and efficient vaccines. As described in the latest blueprint published by the European Commission, live attenuated viruses (LAVs) are the most plausible ASF-vaccine option in the short term [10]. The only data indicating protection from challenge infection available today against the pandemic ASFV strain, currently circulating in Asia and Europe, come from the use of naturally attenuated virus strains [11,12] or recombinant LAVs [13–18]. Despite their protective abilities, LAVs have to improve their safety and DIVA capabilities to be commercially viable in ASF-free areas. While recognizing the key role that future licensed ASF LAVs will play to control ASF in endemic and infected areas and/or in emergencies, it is indispensable to continue our research on safer ASF vaccine alternatives for global use. The presence of more than 150 open reading frames (ORFs) in the ASFV genome [9,19,20], together with the lack of protection correlates, hampers the design of rational subunit vac- cine formulations. As a good example, and even though the crucial role of CD8+ T-cells in protection against ASFV has been established [21], the relevant antigens specifically stimu- lating this cell subset and inducing protective responses have not been fully determined yet. Thus, subunit vaccine formulations used in experimental setups have only conferred partial protection against ASF, and, unfortunately, these results have not been reproduced against the Georgia2007/1 pandemic virus currently circulating [22]. Gaining knowledge on this critical gap is essential to allow the rational design of future subunit vaccines. Due to the failure of previous formulations using DNA vaccination alone against Georgia2007/1 and aiming to increase the chances to discover new T-cell determinants with protective potential against Georgia2007/1, we decided to perform two independent in vivo experi- ments following a heterologous prime–boost regime. Therefore, here we first showed that DNA immunization of pigs with a cocktail of 15 plasmids encoding one ASFV protein eac, enhanced the protection afforded by a suboptimal dose of BA71∆CD2 [14] and confirmed M448R as the main target for the T-cells induced. The proteins were selected based on SLAI binding peptides identified using porcine alveolar macrophages (PAMs) infected with ASFV in vitro. A second immunization experiment was performed by priming pigs with a mix of pCMV-Ub-M448R and pCMV-Ub-MGF505-7R only, encoding M488R and MGF505-7R, previously identified as commonly recognized by CD8+ T-cells [23], to identify their protective potential. We finally discuss the possibilities of these methodologies for antigen discovery and designing future subunit vaccines. 2. Materials and Methods 2.1. Cells Porcine primary cells were collected from ear tissue samples and fibroblast mono- layers were obtained following previously described protocols [24]. Porcine peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Histopaque-1077 density gradient solution (Sigma-Aldrich, Saint Louis, MO, USA) and maintained in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated FBS (GE HealthCare, Chicago, IL, USA), 100 IU/mL of penicillin (Invitrogen, Carlsbad, CA, USA), 100 µg/mL of streptomycin (Invitrogen) and 2 mM L- Vaccines 2021, 9, 508 3 of 16 glutamine (Invitrogen). Fifty micromolar β-mercaptoethanol (Sigma-Aldrich, Saint Louis, MO, USA) was added to the medium to help maintain reducing conditions during the ELISpot assays. Rabbit kidney epithelial RK13 cells were cultured in DMEM supplemented with 10% FBS, 100 IU/mL of penicillin, 100 µg/mL of streptomycin and 2 mM L-glutamine. 2.2. African Swine Fever Virus Strains BA71∆CD2 is a LAV lacking the CD2v gene (EP402R), obtained by homologous recombination from the parental virulent BA71 ASFV strain [14]. Immunization with BA71∆CD2 protects pigs against homologous and heterologous ASFV lethal challenge in a dose dependent manner. Georgia2007/1 is the pandemic virulent strain of ASFV currently circulating in Asia and Europe and was kindly provided by Dr. Linda Dixon (The Pirbright Research Institute, UK). 2.3. Identification of SLAI-Bound Peptides First, 5 × 106 PAMs/well were seeded in a 6-well plate for ASFV infection, using a multiplicity of infection (MOI) of 0.1. Cells were harvested by scrapping when cytopathic effect was evident. Cells were sedimented by centrifugation and obtained cell pellets were frozen at −80 ◦ C until further use for affinity purification of SLAI molecules followed by liquid chromatography coupled to mass spectrometry (LC-MS/MS) as previously described [23]. 2.4. Plasmids Encoding ASFV Genes Fifteen Georgia2007/1 ORFs encoding B475L, B602L, CP2475L (partial), D339L, DP238L, EP424R, H339R, I226R, I243L, I73R, I9R, K145R, M448R, MGF505-1R and MGF505-3R (Gen- Bank accession number FR682468) were individually cloned into the pCMV plasmid (Clon- tech Laboratories, Inc., Mountain View, CA, USA). The plasmid pCMV-Ub-MGF505-7R was previously used to define MGF505-7R as an ASFV antigen recognized by ASFV-specific CD8+ T-cells [23]. Each ORF was cloned in frame with ubiquitin at the N-terminus with the aim to improve proteasome degradation and subsequent CD8+ T-cell induction [25]. In addition, a FLAG-tag sequence was located at the C-terminal end to facilitate the de- tection of protein expression [26]. Protein expression was analyzed by anti-FLAG-tag immunofluorescence in transfected RK13 cells. Transfection of RK13 cells was performed using Lipofectamine 3000 transfection kit (Invitrogen) according to the manufacturers’ instructions. After paraformaldehyde fixation and subsequent Tween-20 permeabilization, cell monolayers were incubated with AlexaFluor 488-conjugated anti-FLAG-tag mono- clonal antibody (MA1-142-A488, Invitrogen) diluted 1:100. Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) was used for nucleus staining. Cells were finally examined by fluorescence microscopy. 2.5. In Vivo Experimental Design Two independent experiments were performed using groups of five four-week-old male piglets (Landrace × Large White), housed in experimental boxes of the biosafety level 3 facilities at IRTA-CReSA (Barcelona, Spain). After 7 days of acclimatization, all pigs were immunized using a heterologous prime–boost regime, priming twice with plasmid DNA encoding ASFV proteins (DNA priming) and subsequently boosted with 103 plaque- forming units (PFU) of BA71∆CD2, a recombinant live attenuated vaccine dose previously described as partially protective [14]. For DNA priming, pigs were immunized with either the empty pCMV-Ub plasmid (negative control) or a mixture of the corresponding recom- binant plasmids. Pigs were immunized twice with 0.6 mg of total endotoxin-free DNA (Qiagen, Hilden, Germany) in 1.5 mL saline, two weeks apart, following the previously described protocol [27]. Two weeks after the second DNA immunization, all pigs were intramuscularly inoculated (heterologous boost) with 103 PFU of BA71∆CD2. Finally, pigs were challenged intramuscularly with a lethal dose of 103 gene equivalent copies (GEC) of Georgia2007/1, three weeks after last immunization (Figure 1). Blood samples and nasal Vaccines 2021, 9, x FOR PEER REVIEW 4 of 18 described protocol [27]. Two weeks after the second DNA immunization, all pigs were Vaccines 2021, 9, 508 intramuscularly inoculated (heterologous boost) with 103 PFU of BA71ΔCD2. Finally,4pigs of 16 were challenged intramuscularly with a lethal dose of 103 gene equivalent copies (GEC) of Georgia2007/1, three weeks after last immunization (Figure 1). Blood samples and nasal swabs were taken at different time points after: DNA primary vaccination (0, 4 and 7 days swabs post were taken primary at different vaccination timeBA71ΔCD2 (dpp)), points after:boost DNA(0,primary 4, 7 andvaccination (0, booster 14 days post 4 and 7 days vac‐ post primary vaccination (dpp)), BA71∆CD2 boost (0, 4, 7 and 14 days post booster cination (dpb)) and Georgia2007/1 challenge (0, 4, 7, 14 and 21 days postchallenge infec‐ vacci- nation tion (dpb))Rectal (dpc)). and Georgia2007/1 challenge temperatures were (0, 4, 7, daily, monitored 14 andas 21 well days as postchallenge ASF typicalinfection clinical (dpc)). Rectal temperatures were monitored daily, as well as ASF typical clinical signs, signs, including behavior, body condition (prominence of vertebrae and ribs), cyanosis, including behavior, body condition (prominence of vertebrae and ribs), cyanosis, digestive digestive and respiratory signs. Each parameter was scored from 0 to 3 according to the and respiratory signs. Each parameter was scored from 0 to 3 according to the severity (0: severity (0: normal; 1: mild; 2: moderate; 3: severe). Post‐mortem examinations were car‐ normal; 1: mild; 2: moderate; 3: severe). Post-mortem examinations were carried out to ried out to confirm or discard the presence of ASF typical pathological lesions [28]. Animal confirm or discard the presence of ASF typical pathological lesions [28]. Animal care and care and procedures were carried out in accordance with the guidelines of the Good Ex‐ procedures were carried out in accordance with the guidelines of the Good Experimental perimental Practices and under the supervision of the Commission of Animal Experimen‐ Practices and under the supervision of the Commission of Animal Experimentation of tation of Generalitat de Catalunya (approval code CEA‐OH/9212/2). As mentioned above, Generalitat de Catalunya (approval code CEA-OH/9212/2). As mentioned above, two two independent in vivo experiments were performed priming pigs with different plas‐ independent in vivo experiments were performed priming pigs with different plasmid mid compositions. In the first experiment, pigs were primed with the cocktail of 15 plas‐ compositions. In the first experiment, pigs were primed with the cocktail of 15 plasmids, mids, and, in the second experiment, pigs were primed with a mix of pCMV‐Ub‐M448R and, in the second experiment, pigs were primed with a mix of pCMV-Ub-M448R and and pCMV‐Ub‐MGF505‐7R, pCMV-Ub-MGF505-7R, following following the procedure the procedure described described above. above. Figure 1. Figure Schematic representation 1. Schematic representation of of the the in in vivo vivo experimental experimental designs. designs. The The two two in in vivo vivo experiments experiments performed performed in in this this study followed an identical scheme but priming with different study followed an identical scheme but priming with different plasmid combinations: either 15 clones in Experiment either 15 clones in Experiment 1 or 1 or the the combination combination ofof two two plasmids plasmids (pCMV‐Ub‐M448R (pCMV-Ub-M448R ++ pCMV‐Ub‐MGF505‐7R) pCMV-Ub-MGF505-7R) in Experiment 2. Groups Groups ofof five five pigs pigs were were primed primed twice twice two weeks apart using the indicated DNA plasmid mixes and boosted with 10 1033 PFU of BA71∆CD2. Three Three weeks weeks after the boost, pigs were challenged with a lethal dose of Georgia2007/1. Samples were taken after the boost, pigs were challenged with a lethal dose of Georgia2007/1. Samples were taken at at different different days days post post priming priming (dpp), (dpp), post post boost boost (dpb) (dpb) or or post post challenge challenge (dpc). (dpc). 2.6. 2.6. Analytical AnalyticalAssays Assays DNA was obtained from DNA was obtained fromsera seraand andnasal nasalswab-PBS swab‐PBS suspensions suspensions (NucleoSpin (NucleoSpin Blood Blood kit; kit; Macherey‐Nagel, Düren, German) and ASFV‐specific DNA Macherey-Nagel, Düren, German) and ASFV-specific DNA was quantified by real-time was quantified by real‐ time PCR PCR (qPCR), (qPCR), usingusing methodsmethods previously previously described described in ourinlaboratory our laboratory [29]. Results [29]. Results were were expressed expressed as logas10 log 10 numbers numbers of of GEC GEC per per mL mL of of sera sera or or nasal nasal swab‐PBS swab-PBS suspension. suspension. The The limitlimit of detection of detection of theofassay the assay was established at 103atGEC/mL. was established 103 GEC/mL. For comparative For comparative pur‐ purposes, poses, our qPCRour technique qPCR technique has previously has previously shown shown more more reproducible reproducible results inresults in sera sera than than in whole in whole blood. Therefore, virus in sera and no viremia was measured, blood. Therefore, virus in sera and no viremia was measured, being aware that the amount being aware that the amount of virus of virus present present in blood in blood would would1be be around logaround higher1inlog higher in magnitude. magnitude. ASFV‐specific immunoglobulinGG(IgG) ASFV-specific immunoglobulin (IgG)in in pigpig serasera werewere detected detected by thebyOIE-approved the OIE‐ap‐ proved indirectindirect ELISA ELISA assay based assay based on theonusethe ofuse of soluble soluble extracts extracts fromfrom ASFV‐infected ASFV-infected cellscells [30]. Plates [30]. were were Plates read at a wavelength read of 450 nm at a wavelength andnm of 450 results andfrom individual results pigs werepigs from individual expressed were as optical density expressed as optical(OD) values. density (OD) values. ASFV specific T-cell responses ASFV specific T‐cell responses were were assessed assessed forfor each each individual individual pig pig at at the the indicated indicated times after times after immunization immunization and after Georgia2007/1 Georgia2007/1challenge challenge bybyIFNΥ IFNΥ ELISpot ELISpot assay assay using using PBMCs from immunized pigs as previously reported [29]. As specific stimuli, PBMCs were incubated overnight with 105 PFU BA71∆CD2 per well (multiplicity of infection of 0.2). RPMI and 10 µg/mL phytohemagglutinin-M (PHA-M, Sigma-Aldrich, Saint Louis, MO, USA) were used as negative and positive controls, respectively. The frequency of specific IFNΥ-secreting Vaccines 2021, 9, 508 5 of 16 cells (IFNΥ-SC) represented in the graphs is the mean of two replicates, subtracting the counts in the negative control wells. Three hundred spots/well was considered the limit of our assay resolution (wells with more than 300 spots received a score of 300). When fibroblastoid cells were used as antigen presenting cells (APCs) in the ELISpot assay, the ratio used was one APC to five autologous PBMCs, and plasmid transfection was performed by electroporation using the Neon Transfection System 10 µL Kit (Invitrogen, Carlsbad, CA, USA), as previously described [23]. The number of spots in a control well using fibroblastoid cells transfected with the empty pCMV-Ub plasmid, which never exceeded 10, was subtracted from the specific IFNΥ-SC represented in the graphs. 2.7. Statistical Analysis For transparency, data from individual animals (always groups of five pigs each) are provided. Complementarily, statistical analysis was inferred between groups applying a standard lineal model (SliM) at different time points while there were enough pigs alive. Thus, no statistical analysis could be performed from the time in which control groups counted with only one surviving pig (in both experiments). This analysis was done using RStudio software [31] with tidyverse and survival packages [32,33]. Statistical significance was set at p < 0.05. Sample size is one of the main factors affecting the power of statistical tests, therefore due to lack of enough surviving pigs in the control group (only one out of five in each experiment) analysis were limited. 3. Results 3.1. DNA Immunization with a Cocktail of Plasmids Encoding 15 ASFV Pre-Selected Proteins Improves Protection against Georgia2007/1 Challenge Infection To confirm the potential of our immunopeptidomic assays to identify potentially protective ASFV antigens, we infected a randomly selected PAM sample from our cell culture collection with BA71∆CD2, at a MOI of 0.1. Fifty-four hours after infection, cells were lysed, SLAI-peptide complexes were immunoprecipitated, the SLAI-bound peptides were eluted and subjected to LC-MS/MS as recently described [23]. As shown in Table 1, 17 individual ASFV peptides were identified, all of them between 8 and 12 amino acids in length and belonging to 15 different antigens (Table 1). The corresponding ORFs were cloned into the pCMV plasmid, under the control of an immediate early promoter of human cytomegalovirus for eukaryotic expression. Each ORF was cloned in frame with ubiquitin to optimize their SLAI antigen presentation and the induction of specific CD8+ T-cell responses [29,34] and with the FLAG-tag peptide in their C-terminal end to confirm their expression. Thus, before any in vivo experiment, the expression of each protein was tested by transient transfection of RK13 cells and anti-FLAG tag immunofluorescence (Table 1). Table 1. Selection of ASFV ORFs for in vivo immunization studies. Peptides Protein Activity/Similarity Plasmid Anti-FLAG Tag DSFIPKEYSQSI NKKLYEKML B475L Unknown pCMV-Ub-B475L-Flag + RKQELLTSQEL Major capsid protein p72 KVDEFYYKY B602L pCMV-Ub-B602L-Flag + chaperone ITKTFVNNI p37 (CP2475L/partial) Structural polyprotein pCMV-Ub-P37-Flag + RSKKDFKQI D339L RNA polymerase subunit 7 pCMV-Ub-D339L-Flag + YSEKEKETI DP238L Unknown pCMV-Ub-DP238L-Flag + FTS J-like methyl transferase NKIKLLNEYL EP424R pCMV-Ub-EP424R-Flag + domain NPTIIMEQY H339R Unknown pCMV-Ub-H339R-Flag + KNILNTLMF I226R Unknown pCMV-Ub-I226R-Flag + Vaccines 2021, 9, 508 6 of 16 Table 1. Cont. Peptides Protein Activity/Similarity Plasmid Anti-FLAG Tag NTILTNKI I243L Transcription factor SII pCMV-Ub-I243L-Flag + TAKNIKVVI I73R Unknown pCMV-Ub-I73R-Flag + YKIYIHSDL I9R Unknown pCMV-Ub-I9R-Flag + YIKTSKQEYL K145R Unknown pCMV-Ub-K145R-Flag + RAKIPAQEI M448R RNA ligase pCMV-Ub-M448R-Flag + YAIHHAPKL MGF505-1R Unknown pCMV-Ub-MGF505-1R-Flag + KKYQHKHIL MGF505-3R Unknown pCMV-Ub-MGF505-3R-Flag + All SLAI peptides identified by MS-based immunopeptidomics using ASFV-infected PAMs are listed in column Peptides, while the ASFV proteins containing the identified peptides and their putative functions are listed in column Protein and Activity/Similarity, respectively. The plasmids encoded each of the selected ASFV ORFs as fusion with ubiquitin at their N-terminus and with a FLAG-tag in the carboxyl terminal end (listed in the column Plasmid). Immunofluorescence assays using an anti-FLAG antibody allowed detecting the expression of the fusion proteins after transient transfection of each plasmid in RK13 (indicated with a + sign). Once the expression of the appropriate protein was confirmed, a group of five pigs was immunized twice two weeks apart with the 15 plasmids cocktail (priming). Five pigs receiv- ing pCMV-Ub served as control group. Two weeks later, all pigs were boosted with 103 PFU of BA71∆CD2, and three weeks later were challenged with a lethal dose of Georgia2007/1 (Figure 1). As expected from previous results using 103 PFU of BA71∆CD2 [14], only one out of the five control pigs (20%) survived the lethal challenge with Georgia2007/1 (Figure 2A). Conversely, three out of five (60%) pigs primed with the 15 recombinant Vaccines 2021, 9, x FOR PEER REVIEW 7 of 18 plasmids and boosted with 103 PFU BA71∆CD2 survived the lethal challenge with Geor- gia2007/1 (Figure 2A). Days post Georgia2007/1 challenge 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 180 181 15 clones 182 183 184 185 186 Control 187 Clinical score 188 0 189 1 2 3 ≥4 Figure Figure Priming 2. 2. pigs Priming with pigs 1515 with plasmids plasmids improves improvesthe protection the protection induced induced byby suboptimal suboptimalBA71∆CD2 BA71ΔCD2 immunization immunization against against lethal Georgia2007/1 lethal Georgia2007/1challenge. challenge.Pigs Pigswere wereimmunized immunized twice twice with either the empty empty pCMV‐Ub pCMV-Ub plasmid plasmid (Control) (Control)or orthe the15 15plasmids plasmidsand and next boosted with 103 PFU BA71∆CD2. next boosted BA71ΔCD2. Two weeks weeks later, later,all allpigs pigswere werechallenged challengedwith witha alethal lethal dosedoseof of Georgia2007/1and Georgia2007/1 and(A) (A)deaths, deaths,(B) (B)ASF-compatible ASF‐compatibleclinical clinicalsigns signsand and(C) (C) rectal rectal temperature temperature from from 1515 plasmids plasmids (top) (top) andand control (bottom) groups were recorded daily. Solid lines represent animals that survived the challenge while control (bottom) groups were recorded daily. Solid lines represent animals that survived the challenge while dashed lines dashed lines symbolize animals that succumbed the challenge. symbolize animals that succumbed the challenge. Clinical signs and rectal temperature matched almost perfectly the viral loads found both in sera (Figure 3A) and nasal swabs (Figure 3B). As expected, pigs succumbing Georgia2007/1 challenge showed detectable ASFV as early as at Day 4 pc. All surviving Vaccines 2021, 9, 508 7 of 16 All pigs that succumbed to Georgia2007/1 challenge suffered typical ASF signs, evident from Days 4–5 post-challenge (pc) (Figure 2B), independently of the group. These included lethargy, general body condition, digestive signs, respiratory signs, cyanosis and fever (Figure 2C). Interestingly, the only control pig surviving Georgia2007/1 challenge, Pig 185, showed high fever during Days 4–9 pc, while immunized pigs only showed brief and milder peaks of fever, with Pig 184 showing no apparent clinical signs typical for ASF. Clinical signs and rectal temperature matched almost perfectly the viral loads found both in sera (Figure 3A) and nasal swabs (Figure 3B). As expected, pigs succumbing Georgia2007/1 challenge showed detectable ASFV as early as at Day 4 pc. All surviving pigs showed a strong reduction of virus in both samples as indicated by decreased GEC. Thus, the only surviving pig from the control group (Pig 185) showed a maximum of 5 Vaccines 2021, 9, x FOR PEER REVIEWGEC of ASFV detectable between Days 7 and 14 pc, while the three surviving pigs from 10 8 of 18 the 15 plasmid group showed a maximum of 104 GEC/mL detectable at Day 7 pc in one sample only (Figure 3A,B). Figure 3. ASFV virus titers in sera and nasal swabs found in pigs after Georgia2007/1 challenge. (A) ASFV titers as indicated Figure 3.found by GEC/mL ASFVinvirus titers(B) sera and innasal sera swabs and nasal swabsbyfound analyzed qPCRinat pigs aftertime different Georgia2007/1 points post challenge. (A) challenge in theASFV titers as 15 plasmids indicated by GEC/mL found in sera and (B) nasal swabs analyzed by qPCR at different time points post challenge in the group (top) and the control group (bottom). Solid lines represent animals that survived the challenge while dashed lines 15 plasmids group (top) and the control group (bottom). Solid lines represent animals that survived the challenge while indicate animals that succumbed to the challenge. dashed lines indicate animals that succumbed to the challenge. 3.2. Immunization with the 15 Plasmids Induces ASFV-Specific T-Cells That Specifically 3.2. Immunization with the 15 Plasmids Induces ASFV‐specific T‐cells that Specifically Recognize M488R Recognize M488R As expected due to the presence of ubiquitin in the N-terminus of the ASFV pro- As expected due to the presence of ubiquitin in the N‐terminus of the ASFV proteins teins [29,34], no antibody responses were detectable after priming with the 15 plasmids [29,34], no antibody responses were detectable after priming with the 15 plasmids (Figure 4A). All pigs seroconverted by Day 14 post‐inoculation with BA71ΔCD2, showing no statistically significant differences between both groups at any time post‐boosting. In addition, a clear boost of antibody titers after Georgia2007/1 challenge was observed in all animals, reaching their maximum titers at the latest time point investigated (Figure 4A). To identify the specificity of the T‐cells recognized by the surviving pigs, PBMCs isolated 21 days after Georgia2007/1 challenge were stimulated in vitro with autologous fibroblasts transfected with each individual recombinant plasmid or a mix containing the Vaccines 2021, 9, 508 15 plasmids, and the specific IFNΥ secretion was detected by ELISpot (Figure 8 of 4C). 16 Interestingly, only the fibroblasts transfected with pCMV‐Ub‐M448R induced significant IFNΥ responses in all four surviving pigs and the response was similar in magnitude to that obtained (Figure 4A). All with fibroblasts transfected pigs seroconverted by Day 14with the mix of the post-inoculation 15 BA71∆CD2, with recombinantshowing plasmids (Figure 4C). Interestingly, M448R was also recognized by PBMCs from control no statistically significant differences between both groups at any time post-boosting.pigs, In indicating addition, thatboost a clear specific T‐cells against of antibody thisGeorgia2007/1 titers after antigen are also induced challenge wasafter BA71∆CD2 observed in all inoculation animals, without reaching theirthe need of atiters maximum DNAatpriming. the latest time point investigated (Figure 4A). Figure 4. M488R is frequently recognized by ASFV-specific T-cells. (A) ASFV-specific antibodies (total IgG) were measured Figure 4. M488R is frequently recognized by ASFV‐specific T‐cells. (A) ASFV‐specific antibodies (total IgG) were measured byby ELISA, expressing ELISA, expressingthe the results as OD results as values at a wavelength OD values of 450of at a wavelength nm.450Solid nm.lines Solidrepresent the 15-ASFV lines represent plasmidsplasmids the 15‐ASFV group and dashed group andlines represent dashed the control lines represent thegroup. control(B) ASFV-specific group. T-cell responses (B) ASFV‐specific were assessed T‐cell responses by IFNΥby were assessed ELISpot using IFNΥ ELISpot PBMCs using isolated PBMCsatisolated differentattime points:time different afterpoints: DNA prime (14 dpp), after DNA after(14 prime BA71∆CD2 dpp), afterboost (21 dpb) and BA71∆CD2 after boost (21Georgia2007/1 dpb) and after challenge (21 dpc). Georgia2007/1 (C) IFNΥ challenge (21ELISpot dpc). (C)using IFNΥPBMCs ELISpotfrom usingsurviving PBMCs fromanimals as effector surviving cells animals asand autologous effector cells andfibroblasts autologous fibroblasts transfected transfected with with pCMV‐Ub‐M448R pCMV-Ub-M448R or the 15 plasmidsor the 15 plasmids as specific as †specific stimuli. stimuli. indicates animals† indicates succumbing animals to ASFVsuccumbing challenge. to ASFV challenge. Immunization with the 15 plasmids induced statistically significant ASFV-specific IFNΥ responses, 3.3. DNA detectable Immunization 14pCMV‐Ub‐M448R with days after the second and plasmid injection (Figure pCMV‐Ub‐MGF505‐7R 4B). By Day Improves 21Protection pb (after against BA71∆CD2 boosting), all pigs showed Georgia2007/1 Lethal Challenge indistinguishable ASFV-specific T-cell responses, from both the 15 plasmids and the control group. Interestingly, the two pigs from Given the promiscuous recognition of M448R by ASFV‐specific T‐cells, we decided the 15 plasmids group that did not survive Georgia2007/1 challenge showed the lowest to test the protective potential of a simple DNA formulation based on two plasmids: level of antibodies and ASFV-specific T response at the time of Georgia2007/1 challenge pCMV‐Ub‐M488R and pCMV‐Ub‐MGF505‐7R, recently described in vitro as an ASFV (Figure+ 4A,B). Surviving pigs did show very strong cellular responses prior euthanasia. CD8 T‐cell antigen [23], following the immunization strategy described before (Figure 1). To identify the specificity of the T-cells recognized by the surviving pigs, PBMCs isolated 21 days after Georgia2007/1 challenge were stimulated in vitro with autologous fibroblasts transfected with each individual recombinant plasmid or a mix containing the 15 plasmids, and the specific IFNΥ secretion was detected by ELISpot (Figure 4C). Interestingly, only the fibroblasts transfected with pCMV-Ub-M448R induced significant IFNΥ responses in all four surviving pigs and the response was similar in magnitude to that obtained with fibroblasts transfected with the mix of the 15 recombinant plasmids Vaccines 2021, 9, 508 9 of 16 (Figure 4C). Interestingly, M448R was also recognized by PBMCs from control pigs, indicat- ing that specific T-cells against this antigen are also induced after BA71∆CD2 inoculation without the need of a DNA priming. 3.3. DNA Immunization with pCMV-Ub-M448R and pCMV-Ub-MGF505-7R Improves Protection against Georgia2007/1 Lethal Challenge Vaccines 2021, 9, x FOR PEER REVIEW Given the promiscuous recognition of M448R by ASFV-specific T-cells, we decided to18 10 of test the protective potential of a simple DNA formulation based on two plasmids: pCMV- Ub-M488R and pCMV-Ub-MGF505-7R, recently described in vitro as an ASFV CD8+ T-cell antigen [23], following the immunization strategy described before (Figure 1). Once again, Once again, only one out of the five control pigs (primed twice with pCMV‐Ub) survived only one out of the five control pigs (primed twice with pCMV-Ub) survived Georgia2007/1 Georgia2007/1 lethal challenge. On the contrary, three out of the five pigs primed with lethal challenge. On the contrary, three out of the five pigs primed with pCMV-Ub-M488R pCMV‐Ub‐M488R and pCMV‐Ub‐MGF505‐7R survived (Figure 5A). In this occasion, two and pCMV-Ub-MGF505-7R survived (Figure 5A). In this occasion, two out of the three out of the three surviving pigs from the immunized group showed no clinical signs surviving pigs from the immunized group showed no clinical signs compatible with ASF compatible with ASF (Figure 5B), with Pig 89 showing no fever at any time point investi‐ (Figure 5B), with Pig 89 showing no fever at any time point investigated, while Pig 90 gated, while showed a peakPigof 90 showed fever at thea end peakofofthefever at the study end of5C). (Figure the The study (Figure third 5C). The surviving pigthird in surviving pig in the immunized group (Pig 88) recovered from mild the immunized group (Pig 88) recovered from mild apathy and early fever, resolving the apathy and early fever, resolving infection the infection afterwards. Conversely,afterwards. the onlyConversely, control pigthe only control surviving ASFV pig surviving lethal ASFV challenge lethal challenge (Pig 99) suffered prolonged lethargy starting at Day 9 pc and (Pig 99) suffered prolonged lethargy starting at Day 9 pc and lasting until the end of the trial. lasting until the end of the trial. Moreover, it developed cyanosis in ears and tail, Moreover, it developed cyanosis in ears and tail, even though fever was only detectable at even though fever was the end only detectable of the experiment at the end of5C). (Figure theMatching experiment (Figure fever 5C). Matching and clinical fever and clinical signs, succumbing pigs signs, succumbing showed higher virus pigsGECshowed numbers higher virus in both seraGEC numbers (Figure 6A) andin both nasalsera swabs (Figure (Figure6A)6B), and nasal 3–4 logsswabs above(Figure 6B), 3–4 those found inlogs above pigs. surviving those Only foundPig in surviving 89 showedpigs. Only Pig 89 no detectable showed virus in no detectable serum at any timevirus in infection. after serum at any time after infection. A C % survival B 1 2 3 4 5 Days post Georgia2007/1 challenge 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 86 MGF505-7R 87 M448R + 88 89 90 96 Clinical score 97 0 Control 98 1 99 2 100 3 ≥4 Figure Priming 5. 5. Figure Primingpigspigswith pCMV-Ub-M448R with pCMV‐Ub‐M448R andand pCMV-Ub-MGF505-7R pCMV‐Ub‐MGF505‐7R improves the protection improves afforded the protection by suboptimal afforded by subopti‐ BA71∆CD2 immunization mal BA71ΔCD2 immunizationagainst lethallethal against Georgia2007/1 challenge. Georgia2007/1 PigsPigs challenge. were immunized were immunized twice with twice either with thethe either empty empty pCMV‐Ub plasmid (Control) or pCMV‐Ub‐M488R + pCMV‐Ub‐MGF505‐7R and next boosted pCMV-Ub plasmid (Control) or pCMV-Ub-M488R + pCMV-Ub-MGF505-7R and next boosted with 10 PFU BA71∆CD2. with 3 10 3 PFU BA71ΔCD2. Two Two weeks weeks later, later, allallpigs pigswere werechallenged challengedwith with aa lethal lethal dose dose of ofGeorgia2007/1 Georgia2007/1 and (A) and deaths, (A) (B)(B) deaths, ASF typical ASF clinical typical signs clinical and (C) rectal temperature from pCMV‐Ub‐M488R + pCMV‐Ub‐MGF505‐7R (top) and control signs and (C) rectal temperature from pCMV-Ub-M488R + pCMV-Ub-MGF505-7R (top) and control (bottom) groups (bottom) groups were rec‐ orded daily. Solid lines represent animals that survived the challenge while dashed lines symbolize animals that suc‐ were recorded daily. Solid lines represent animals that survived the challenge while dashed lines symbolize animals that cumbed to the challenge. succumbed to the challenge. Vaccines 2021, 9, x508 Vaccines FOR PEER REVIEW 1110ofof 18 16 Figure 6. Virus DNA titers found in sera and nasal swabs of pigs after Georgia2007/1 challenge. Figure 6. Virus DNA titers found in sera and nasal swabs of pigs after Georgia2007/1 challenge. (A) ASFV GEC titers found in sera and (B) nasal swabs at different times post Georgia2007/1 (A) ASFV GEC titers found in sera and (B) nasal swabs at different times post Georgia2007/1 chal‐ challenge detected by qPCR in the pCMV-Ub-M448R and pCMV-Ub-MGF505-7R primed group (top) lenge detected by qPCR in the pCMV‐Ub‐M448R and pCMV‐Ub‐MGF505‐7R primed group (top) andthe and thecontrol controlgroup group(bottom). (bottom).Solid Solid lines lines represent represent animals animals thatthat survived survived the the challenge challenge while while dashedlines dashed linessymbolize symbolize animals animals that that succumbed succumbed to to the the challenge. challenge. 3.4. M448R and MGF505-7R Are Frequently Recognized by ASFV-Specific T-Cells 3.4. M448R and MGF505‐7R are Frequently Recognized by ASFV‐Specific T‐Cells Administration of the pCMV-Ub-M448R and pCMV-Ub-MGF505-7R plasmids did not Administration of the pCMV‐Ub‐M448R and pCMV‐Ub‐MGF505‐7R plasmids did induce any detectable ASFV-specific antibody response in the IgG ELISA (Figure 7A) due to not the induce presenceanyof detectable ubiquitin inASFV‐specific their N-terminusantibody response [29,34]. in theall Conversely, IgG ELISA pigs (Figure but one (Pig 7A) 100) due to the presence of ubiquitin in their N‐terminus [29,34]. Conversely, showed similar antibody responses by day 21 post BA71∆CD2 boosting. Surviving all pigs but pigs one (Pig 100) maximum showed showed similar antibody antibody responses titers after by day 21 challenge, Georgia2007/1 post BA71∆CD2 boosting. Surviv‐ albeit protection did not ing pigs showed maximum antibody titers after Georgia2007/1 challenge, perfectly correlate with the levels of antibodies existing at the time of challenge albeit(Figure protection 7A). did not perfectly Priming withcorrelate with the levels pCMV-Ub-M448R andofpCMV-Ub-MGF505-7R antibodies existing at the time of induced challenge a significant (Figure number7A).of T-cells that specifically recognized ASFV in vitro. The number of T-cells de- tectable after BA71∆CD2 boost dramatically increased in DNA primed pigs, and also in control pigs, reaching similar levels, with the exception of Pig 100 that showed no specific T-cell responses (Figure 7B). Interestingly, specific IFNΥ responses against M448R and MGF505-7R were detectable in DNA primed pigs before BA71∆CD2 boost, and in both groups of animals after boosting with BA71∆CD2 (Figure 7C, top and bottom). After Georgia2007/1 challenge, surviving animals showed high numbers of T-cells capable to specifically recognize ASFV (Figure 7B), and also the M448R and MGF505-7R ASFV proteins (Figure 7C). Vaccines 2021, Vaccines 2021, 9, 9, 508 x FOR PEER REVIEW 12 11 of 18 of 16 Figure 7. M488R and MGF505-7R are frequently recognized by specific T-cells. (A) ASFV-specific antibodies (total IgG) Figure 7. M488R and MGF505‐7R are frequently recognized by specific T‐cells. (A) ASFV‐specific antibodies (total IgG) were were measured measuredbybyELISA, ELISA,expressing expressingthe theresults resultsasasOD ODvalues valuesatataawavelength wavelengthofof450 450nm. nm.Solid Solidlines linesrepresent representthe theM448R M448R+ MGF505-7R + MGF505‐7R group and dashed lines represent the control group. ASFV‐specific T‐cell responses were assessed by IFNΥ group and dashed lines represent the control group. ASFV-specific T-cell responses were assessed by IFNΥ ELISpot using as stimulus (B) ASFV or (C) (C) autologous autologous fibroblasts fibroblasts transfected transfected with with either either pCMV‐Ub‐M448R pCMV-Ub-M448R or or pCMV‐Ub‐ pCMV-Ub- MGF505-7R MGF505‐7R at different different time time points: points: after after DNA DNA prime prime (14 (14 dpp), dpp), after afterBA71∆CD2 BA71∆CD2 boost boost (21 (21 dpb) dpb)andandafter afterGeorgia2007/1 Georgia2007/1 challenge (21 dpc). Data from the M448R+MGF505-7R M448R+MGF505‐7R primed primed group group and and control control group group are represented at the top and bottom, respectively. bottom, respectively. †† indicates indicates animals animals succumbing succumbing to to ASFV ASFV challenge. challenge. Priming with pCMV‐Ub‐M448R and pCMV‐Ub‐MGF505‐7R induced a significant 4. Discussion numberTheofsocioeconomic T‐cells that specifically impact of recognized ASF ranks ASFV this in vitro.swine lethal The number diseaseofasT‐cells detect‐ the number able after BA71∆CD2 boost dramatically increased in DNA primed pigs, one challenge for the pig industry worldwide. This relates to animal well-being, trade and also in con‐ trol pigs, reaching similar levels, with the exception of Pig 100 that showed no restriction and consequently availability of food at affordable prices based on pork. As one specific T‐ cell responses of the (Figure 7B).measures, ASF vaccine development has a major priority. Thus most cost-effective Interestingly, far, only LAVs havespecific shownIFNΥsolid responses protectionagainst againstM448R and MGF505‐7R ASFV. Even though mostwere detecta‐ of the times ble in DNA primed pigs before BA71∆CD2 boost, and in both groups protection is limited to homologous protection [13,16,17], LAVs have become essential of animals after boosting with BA71∆CD2 (Figure 7C, top and bottom). After Georgia2007/1 tools to confirm the protective role of innate [35–38] and adaptive immune responses, challenge, surviving with both animals humoralshowed response high numbers [39–41] and of T‐cells capable ASFV-specific CD8 to+specifically T-cells [21]recognize ASFV playing crucial (Figure roles in 7B), and alsoInthe protection. M448R this regard,and MGF505‐7RLAVs recombinant ASFVare proteins (Figure the most 7C). candidates advanced against the pandemic virus [14–17]. Although still pending approval from the authorized 4. Discussion agencies, LAVs candidates should contribute in the near future to control governmental ASF The in endemic areas and socioeconomic in emergencies. impact of ASF ranks However, it swine this lethal is difficult to envision disease the useone as the number of LAVs in ASF-free challenge for the areas in the short pig industry term, overall worldwide. This taking relatesinto accountwell‐being, to animal the non-vaccination trade re‐ policy applied striction against other and consequently diseases of availability of food obliged declaration at affordable to the prices OIEon based inpork. disease-free As one countries. of the mostOn the other hand, cost‐effective subunit measures, vaccines ASF vaccineagainst ASF arehas development notaa major utopia, but needing priority. Thus extra research to understand the disease and subsequent development far, only LAVs have shown solid protection against ASFV. Even though most of the times efforts [22,42], including discovery protection is limitedoftoprotective homologous antigens. protection [13,16,17], LAVs have become essential tools to confirm the protective role of innate [35–38] and adaptive immune responses, with Vaccines 2021, 9, 508 12 of 16 Subunit vaccine formulations used in experimental setups have only conferred partial protection against ASF [29,34,43]. Unfortunately, these results have not been reproduced against the Georgia2007/1 pandemic virus currently circulating [22,44,45]. Confirming this reality, preliminary work perform in our laboratory using DNA immunization as a tool and CD2v as vaccine target failed to confer any protection against Georgia2007/1 challenge (unpublished results), while the same formulation conferred partial protection against E75, a genotype I ASFV strain isolated in Spain in the 1970s [34]. Differences in the amino acid sequence of CD2v between these two strains might partially explain the different extent of protection. Thus, the two CD8+ T-cell determinants identified in the E75 strain [34] are absent in the CD2v sequence of the Georgia2007/1 ASFV. Nonetheless, many other reasons might contribute to this outcome, including the differential degree of virulence due to a possible different biology observed for these two ASFV strains. Similar results have also been described for LAV prototypes, showing different protection abilities depending on the ASFV strain used for vaccine designing and for experimental challenge [46,47]. Thus far, only complex formulations encoding multiple ASFV antigens have shown some protective efficacy against experimental lethal Georgia2007/1 challenge, but seldom avoiding the death of the animals [45,48]. Due to the failure of previous formulations using DNA vaccination alone against Geogia2007/1 and aiming to increase the chances to discover new T-cell determinants with protective potential against Georgia2007/1, here we decided to immunize pigs following a prime–boost regime. Recent efforts performed using a multiple approach that combined in silico predic- tions, immunopeptidomics and in vitro antigen presentation assays allowed identifying a panel of novel Georgia2007/1 antigens commonly recognized by CD8+ T-cells from surviving pigs [23]. Here, we present parallel studies performed in our laboratory confirm- ing immunopeptidomics as a consistent approach not only to identify ASFV CD8+ T-cell determinants in vitro, but also to discover novel ASFV antigens with protective potential. Taking into account the failure of our experimental DNA vaccines based on CD2v, here we implemented a heterologous prime–boosting immunization protocol, boosting DNA- immunized pigs with a partially protective dose of 103 PFU of BA71∆CD2 [14] aiming to optimize the protective potential of our DNA vaccine formulations against Georgia2007/1. A similar immunization strategy using vector-expressed ASFV antigens as a prime, fol- lowed by a boost with the naturally attenuated OURT88/3 ASFV isolate was previously published [49]. Nevertheless, that specific study was focused on antibody response, and neither cell-mediated response nor protective potential was evaluated. Our heterologous prime–boost protocols were shown to increase the chances to discover ASFV antigens with protective potential for future vaccine formulations in optimized expression vectors. In line with previous experiences, priming with DNA vaccines encoding ubiquiti- nated antigens induced ASFV-specific cellular responses detectable directly after DNA immunization, while no specific antibody response was observed [29,34]. Unexpectedly, no significant differences were observed in the kinetics or in the levels of ASFV-specific IFNΥ-secreting cells after administration of BA71∆CD2 between DNA-primed and control animals, at least at the time tested (21 dpc) and by using the IFNΥ ELISpot as readout. The consistent improvement in the survival rate of vaccinated animals versus control pigs sug- gests that the T-cell repertoire induced by the DNA priming contributed to a better control of the infection. It must be noted that the two vaccine formulations successfully tested in the in vivo experiments here described provided the same degree of protection (60% of the pigs survived the ASFV lethal challenge). Although the clinical score of the surviving pigs seemed slightly better in the group primed with pCMV-Ub-M448R + pCMV-Ub-MGF505- 7R than with the 15 ASFV clones, the differences have no statistical significance. Moving into the field of speculation, plasmids within the plasmids mix neglected due to the lack of significant responses (data not shown) might have contributed to parallel the protec- tion rates observed when combining pCMV-Ub-M448R and pCMV-Ub-MGF505-7R. The protection capabilities of these two plasmids individually were not tested. Vaccines 2021, 9, 508 13 of 16 Combining the parallel approaches described here and in our previous report [23], we aim to identify as many protective determinants as possible in the Georgia2007/1 proteome. Despite the phenotype of the T-cells secreting IFNΥ has not been confirmed, the use of transfected as APCs, in combination with the ubiquitination strategy, points to a CD8+ T cell phenotype, as recently described for other antigens using a similar technology [23]. The lack of in vitro correlates for ASFV protection complicates providing a definitive explanation of why pigs do or do not survive. In agreement with multiple experiences in our lab and independently of the ASFV strain used for challenge, no perfect correlation existed between protection and the level of antibodies detectable by ELISA or the number of IFNΥ specific T-cells detectable by ELISpot at the time of challenge. On some occasions, this relationship seems to exist, as shown in Figure 4 for the IFNΥ spots found before Georgia2007/1 challenge, but, in many others, pigs with large amounts of specific T-cells and antibodies do not survive ASFV challenge. These results confirm that, together with antigen discovery, extra efforts should be directed at searching for consistent correlates of protection. Solving this gap would not only help rational vaccine design, but also avoid unnecessary suffering to pigs, since up to today no alternative to Georgia2007/1 challenge exists to characterize protective determinants or vaccine prototypes. Proteins M448R and MGF505-7R were identified not only as frequently recognized ASFV-specific T-cell determinants, but also as candidates to be included in future and complex vaccine formulations. Even though M448R remains an uncharacterized protein in ASFV [19], orthologs have been described in other dsDNA viruses [50,51]. Their RNA ligase activity is involved in tRNA repair [52], and hence can facilitate host evasion due to bypassing immune response to damaged RNA associated with virus infection [53,54]. M448R sequence is conserved among ASFV isolates [55], a common feature of metabolic enzymes [56]. MGF505-7R ASFV gene (A528R in the old nomenclature), also conserved among ASFV strains [55,57], has been described to encode an IFN inhibitor protein [58]. While this is the first report showing the immunogenicity of the combinations of both proteins (MGF505-7R and M448R), M448R was previously included in a complex experimental vaccine formulation based on recombinant viral vectors encoding individual ASFV proteins [59]. In this case, immunization of both NIH dd minipigs and outbred pigs with this cocktail induced specific humoral and T-cell response against M448R. However, the M448R-specific response was not uniform in all pigs and, despite a delayed onset of clinical signs and reduced viremia and viral loads in tissues, all pigs died after lethal challenge with OURT88/1 ASFV (genotype I). Further testing of MGF505-7R and M448R, using the appropriate vaccine vectors would definitively shed more light on their protective potential. These and others results obtained thus far using complex subunit vaccine formulations [43,44,48] confirm the complexity of developing safe and efficient subunit vaccines in the future as well as the need of identifying as many protective antigens as possible within the ASFV. We believe that this is just one piece of a puzzle, which will also need further research to select the optimal combination of expression vectors and immune adjuvants if aiming to mimic the solid protection conferred by LAVs. In addition, a better understanding of protective innate and adaptive immunity is fundamental to optimize in a rational manner vaccine compositions in the future. Author Contributions: Conceptualization and methodology, L.B.-C., V.N., J.C. and F.R.; investigation, L.B.-C., E.L., M.J.N. and S.P.-P.; formal analysis, M.B.-F.; resources, M.L.S.; writing and visualization— original draft preparation, L.B.-C. and F.R.; and supervision and project administration, F.A., E.M., V.N., J.C. and F.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Departament d’Economia i Coneixement de la Generalitat de Catalunya (Spain), grant number 2015 DI 037. Studies were co-financed by Boehringer Ingelheim Veterinary Research Center (BIVRC) GmbH & Co. KG, the Ministerio de Ciencia e Innovación of Spain (grant numbers AGL2016-78169-C2-1-R and PID2019-107616RB-I00) and Red de Investigación en Sanidad Animal (RISA). Vaccines 2021, 9, 508 14 of 16 Institutional Review Board Statement: The in vivo studies were conducted according to the guide- lines of the Declaration of Helsinki and approved by the Institutional Review Board of Generalitat de Catalunya (approval code CEA-OH/9212/2). Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. 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