viruses Article Deletion Mutants of the Attenuated Recombinant ASF Virus, BA71∆CD2, Show Decreased Vaccine Efficacy Elisabeth Lopez 1 , Laia Bosch-Camós 1 , Elizabeth Ramirez-Medina 2 , Elizabeth Vuono 2 , Maria Jesus Navas 1 , Marta Muñoz 1 , Francesc Accensi 1,3 , Jinya Zhang 1 , Uxia Alonso 1 , Jordi Argilaguet 1 , Maria Luisa Salas 4 , Nikolay Anachkov 5 , Douglas P. Gladue 2,† , Manuel V. Borca 2,† , Sonia Pina-Pedrero 1, *,† and Fernando Rodriguez 1, *,† 1 IRTA, Centre de Recerca en Sanitat Animal (IRTA-CReSA), Campus de la Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain; elisabeth.lopezf@gmail.com (E.L.); Laia.Bosch@irta.cat (L.B.-C.); mariajesus.navas@irta.cat (M.J.N.); Marta.munoz@irta.cat (M.M.); Francesc.accensi@irta.cat (F.A.); jinya.zhang@irta.cat (J.Z.); Uxia.alonso@irta.cat (U.A.); jordi.argilaguet@irta.cat (J.A.) 2 USDA Plum Island Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Greenport, NY 11944, USA; Elizabeth.Ramirez@usda.gov (E.R.-M.); Elizabeth.Vuono@usda.gov (E.V.); Douglas.Gladue@usda.gov (D.P.G.); Manuel.Borca@usda.gov (M.V.B.) 3 Departament de Sanitat i d’Anatomia Animals, Facultat de Veterinària, UAB, 08193 Bellaterra, Spain 4 Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Campus de la Universidad Autònoma de Madrid, 28049 Madrid, Spain; mlsalas@cbm.csic.es 5 Biologics Development, Huvepharma, 3A Nikolay Haytov Street, 1113 Sofia, Bulgaria; n_anachkov@biovet.com  * Correspondence: sonia.pina@irta.cat (S.P.-P.); Fernando.rodriguez@irta.cat (F.R.)  † These authors contributed equally to this work. Citation: Lopez, E.; Bosch-Camós, L.; Ramirez-Medina, E.; Vuono, E.; Abstract: African swine fever (ASF) has become the major threat to the global swine industry. Lack Navas, M.J.; Muñoz, M.; Accensi, F.; of available commercial vaccines complicates the implementation of global control strategies. So Zhang, J.; Alonso, U.; Argilaguet, J.; far, only live attenuated ASF viruses (ASFV) have demonstrated solid protection efficacy at the et al. Deletion Mutants of the experimental level. The implementation of molecular techniques has allowed the generation of a Attenuated Recombinant ASF Virus, collection of deletion mutants lacking ASFV-specific virulence factors, some of them with promising BA71∆CD2, Show Decreased Vaccine potential as vaccine candidates against the pandemic genotype II ASFV strain currently circulating in Efficacy. Viruses 2021, 13, 1678. https://doi.org/10.3390/v13091678 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 Academic Editors: Chao-Nan Lin and recombinant live attenuated virus (LAV) lacking the ASFV CD2v gene (encoding β-glucuronidase as a Peck Toung Ooi 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 Received: 13 July 2021 gene instead of CD2v, and two double recombinants lacking CD2v and either the lectin gene (EP153R) Accepted: 6 August 2021 or the uridine kinase (UK) gene (DP96R). Comparative in vivo experiments using BA71∆CD2f, Published: 25 August 2021 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 Publisher’s Note: MDPI stays neutral improve safety. Our results additionally confirm ASFV challenge as the only available method today with regard to jurisdictional claims in to evaluate the protective efficacy of any experimental vaccine. We believe that understanding the published maps and institutional affil- fine equilibrium between attenuation and inducing protection in vivo deserves further study and iations. might contribute to more rational vaccine designs in the future. Keywords: African swine fever (ASF); African swine fever virus (ASFV); live attenuated virus (LAV); vaccine; virulence factor; double mutant; protection Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons Attribution (CC BY) license (https:// Lack of commercially available vaccines complicates the control of African swine creativecommons.org/licenses/by/ fever (ASF), today’s number one threat to the global swine industry [1]. The complexity 4.0/). of African swine fever virus (ASFV), the causative agent of ASF, together with the limited Viruses 2021, 13, 1678. https://doi.org/10.3390/v13091678 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 1678 2 of 13 resources historically invested in this disease, has delayed vaccine development. This situation has dramatically changed with the last re-entrance of the virus in Europe in 2007 and the subsequent spread to the West, reaching the European Union in 2014, and to the East, reaching China in 2018. It has been shown that classically inactivated vaccines for ASF are ineffective, independent of the adjuvant tested [2]. To date, all subunit vaccines that have been reported have failed at inducing solid protection against the pandemic ASFV (Georgia2007/1), despite being the ideal choice in the long term if one becomes available [3]. So far, only ASF live attenuated viruses (LAVs) have conferred solid pro- tection against experimental challenge with homologous viruses, with both antibodies and T cells, playing important roles in the protection afforded [4,5]. These studies have been performed with either natural attenuated ASFV isolates or with recombinant LAVs obtained by specific deletions of virulence-associated genes from the homologous virulent virus [6–11]. Additionally, our previous studies have demonstrated the cross-protective ability of BA71∆CD2, a deletion mutant lacking the CD2v (encoding β-glucuronidase as a reporter gene) from the genotype I virulent BA71 ASFV strain, to confer protection against both parental BA71 and genotype II Georgia2007/1 experimental challenges [12]. Despite its protective potential and the ability to grow in stable cell cultures (Cos-1 cells) without genetic changes, a small percentage of BA71∆CD2-immunized pigs showed low, but detectable, amounts of ASFV DNA in sera [12]. Aiming to reduce the residual viral load observed with BA71∆CD2 and increase the safety of our vaccine prototype, three new recombinant viruses were generated using the same methodology and substituting the ASFV ORFs with fluorescent markers: the single mutant BA71∆CD2f, this time containing the fluorescent mCherry marker instead of CD2v, and two double recombinants lacking CD2v and a second virulence-associated gene, either DP96R, encoding the ASFV uridine kinase gene [8], or EP153R, encoding the ASFV lectin [13]. Once generated, the two double mutants, BA71∆CD2DP96R and BA71∆CD2EP153R, were used in a comparative in vivo immunization study with their parental fluorescent counterpart, BA71∆CD2f, whereas the control group of the whole experiment was immunized with PBS. Finally, all pigs were challenged with the pandemic Georgia2007/1 strain. The specific antibody and T-cell re- sponses induced, together with the presence of ASFV in pig sera before and after challenge, determined that the additional deletion of DP96R or EP153R from BA71∆CD2f did not improve the safety of the vaccine, but instead reduced the protective potential against Georgia2007/1 challenge. These studies confirm previous results obtained with other recombinant LAVs designed using genotype II and I ASFV strains as templates [14–18]. The lack of exact correlation between in vitro assays and protection in swine confirms ASFV challenge as the only valid method to accurately evaluate the protective efficacy of any experimental vaccine approach. Understanding the mechanisms governing the efficacy of LAVs might become essential to design safer vaccine approaches against ASF in the future. 2. Methods 2.1. Cells and Viruses Porcine alveolar macrophages (PAMs) were obtained by successive bronchoalveolar lung lavages with PBS. Macrophage cell culture was performed with RPMI 1640 medium (Life Technologies, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (HyClone, GE Healthcare, Chicago, IL, USA). The recombinant viruses were purified by limited dilution on PAMs. The virulent isolate Georgia2007/1, used for challenge purposes, was kindly provided by Dr. Linda Dixon (The Pirbright Institute, Ash Road, Pirbright, Surrey GU24 0NF, UK) and titrated in porcine macrophages. 2.2. Generation of the Recombinant Deletion Mutants and Comparative In Vitro Growth Curves For comparative purposes, we first generated a fluorescent BA71∆CD2f by homolo- gous recombination (Figure 1A), following a similar procedure described before [19]. The right and left genome arms of the CD2 locus (approximately 700–800 bp in length) were p72 late promotor. Macrophage cell cultures were infected with the parental virus BA71∆CD2 (containing a β-glucuronidase, encoded by the GusA gene, as a reporter gene) and transfected with the p72mCherry∆CD2 recombination transfer vector. By homolo- gous recombination, the GusA gene was exchanged by the mCherry reporter gene, gen- Viruses 2021, 13, 1678 erating the recombinant BA71∆CD2f. 3 of 13 Next, we generated the recombinants BA71∆CD2DP96R and BA71∆CD2EP153R, us- ing BA71∆CD2f as the parental virus and p72eGFP∆DP96R or p72eGFP∆EP153R as trans- fer vectors, respectively (Figure 1B). Both transfer vectors harbor the green fluorescent obtained protein (eGFP)by DNA gene synthesis under at Epoch the control Life of the Sciences ASFV (Sugar p72 late geneLand, TX, USA) promoter to construct as a reporter the p72mCherry∆CD2 transfer vector encoding the mCherry reporter gene gene cassette, flanked by the left and right homologous arms of the deleted ORFs to allowunder the ASFV p72 late promotor. Macrophage cell cultures were infected homologous recombination, following the same system described above. with the parental virus BA71∆CD2 (containing a β-glucuronidase, encoded by the GusA gene, as a reporter The recombinant viruses were obtained after successive rounds of limit dilution pu- gene) and transfected with the p72mCherry∆CD2 recombination transfer vector. By homologous rification on macrophage cell cultures, followed by a full genome sequencing to ensure recombination, the GusA gene was exchanged by the mCherry reporter gene, generating the genome integrity and the designed deletions. the recombinant BA71∆CD2f. A B Figure 1. Schematic representation of the recombinant parental virus BA71∆CD2f (A) and the FigureEP153R 1. Schematic representation and DP96R of the gene regions recombinant deleted parentalrecombination by homologous virus BA71∆CD2f (A) and the in recombinant viruses EP153R and DP96R gene regions deleted by homologous recombination in recombinant BA71∆CD2EP153R and BA71∆CD2DP96R (B). Dotted lines indicate the homologous regions viruses between BA71∆CD2EP153R and BA71∆CD2DP96R (B). Dotted lines indicate the homologous regions be- the parental virus and the correspondent transfer plasmid. tween the parental virus and the correspondent transfer plasmid. Next, we generated the recombinants BA71∆CD2DP96R and BA71∆CD2EP153R, using BA71∆CD2f as the parental virus and p72eGFP∆DP96R or p72eGFP∆EP153R as transfer vectors, respectively (Figure 1B). Both transfer vectors harbor the green fluorescent protein (eGFP) gene under the control of the ASFV p72 late gene promoter as a reporter gene cassette, flanked by the left and right homologous arms of the deleted ORFs to allow homologous recombination, following the same system described above. The recombinant viruses were obtained after successive rounds of limit dilution purification on macrophage cell cultures, followed by a full genome sequencing to ensure the genome integrity and the designed deletions. Comparative in vitro growth curves of BA71∆CD2f, BA71∆CD2DP96R and BA71∆CD2EP153R were performed by infecting PAMs at a multiplicity of infection (MOI) of 0.1. After 2 h of adsorption at 37 ◦ C in 5% CO2 , the inocula were removed and replaced with fresh media. The supernatants were harvested at 16, 24 and 48 h post infection (hpi). Virus loads in supernatants were quantified by qPCR following previously described Viruses 2021, 13, 1678 4 of 13 methods [20] and the percentage of infected cells at each time point was followed by flow cytometry, detecting the eGFP or mCherry reporter genes. 2.3. Animals, Hosting and Welfare Six-to-eight-week-old male (Large White × Landrace) pigs, ranging from 15 to 30 kg, were housed in the BSL-3 facilities of Institut de Recerca i Tecnologia Agroalimentària (IRTA)-CReSA (Bellaterra, Spain). Animal experiments were conducted according to animal welfare ethics and protocols approved by the Ethics Commission in Animal Experi- mentation of the Generalitat de Catalunya (code CEA-OH/9212/2, in accordance to the European (Directive 2010/63/EU) and Spanish (Real Decreto 53/2013) regulations under the supervision of the IRTA’s Ethical and Animal Welfare Committee. 2.4. In Vivo Experimental Approach Twenty-four pigs were divided into four groups of six pigs each, without any contact between the groups. After one week of acclimation, each pig was intramuscularly immu- nized once with 1 mL of either PBS (control) or 106 plaque-forming units (PFU) of either BA71∆CD2f, BA71∆CD2EP153R or BA71∆CD2DP96R. Twenty-four days later, all pigs were intramuscularly challenged with 103 gene equiv- alent copies (GEC) of the Georgia2007/1 ASFV virulent strain, equivalent to 103 hemad- sorbing units (HAU). Pigs were bled at days 0, 4, 7, 14 and 24 after immunization (dpi) and after Georgia2007/1 challenge (dpc) to follow both the kinetics of virus load by qPCR and the ASFV-specific antibody and T-cell responses. Rectal temperature and clinical signs were recorded daily, following guides previously published [21]. Post-mortem examinations were carried out to confirm or discard the presence of ASF-compatible pathological lesions. 2.5. Analytical Methods Experimental immunization, clinical observations, immunological assays and virus titration methods were previously described [12,21] and performed as briefly described below. 2.5.1. Antibody Detection by ELISA Antibodies in pig sera were quantified by the OIE-approved indirect ELISA, based on soluble ASFV-infected cell-extract-coated plates, generously provided by Dr. Carmina Gallardo (EU ASF reference laboratory, CISA-INIA, Madrid, Spain). The presence of positive sera was detected using a peroxidase-conjugated anti-pig immunoglobulin G (IgG) at 1:20,000 dilution (Sigma-Aldrich, St. Louis, MO, USA) as secondary antibody and soluble 3,30 ,5,50 -tetramethylbenzidine (TMB) as specific peroxidase substrate (Sigma- Aldrich). Reactions were stopped with 1 N H2 SO4 (Sigma-Aldrich), and the ELISA plates were read at an optical density of 450 nm (OD450 ). 2.5.2. T-Cell Response by ELISPOT Assay The frequency of ASFV-specific interferon gamma-secreting cells (IFNγ-SC) in periph- eral blood mononuclear cells (PBMCs) was analyzed by an enzyme-linked immunosorbent spot (ELISPOT) assay using commercial monoclonal antibody tandems (swine IFNγ; Cy- toset). Briefly, PBMCs were isolated from whole blood by density-gradient centrifugation with Histopaque 1077 (Sigma-Aldrich). For PBMC cultures, RPMI 1640 medium supple- mented with 10% fetal bovine serum (HyClone, GE HealthCare), 50,000 IU penicillin/L (Invitrogen), and 50 mg streptomycin/L (Invitrogen) was used. Trypan blue was used to as- sess cell viability. PBMCs were specifically stimulated for 20 h in vitro with different ASFV isolates at a MOI of 0.2. RPMI, and 10 µg/mL of phytohemaglutinin (PHA, Sigma-Aldrich) was used as the control of the technique. Any sample scoring ≥300 spots/500,000 PBMCs received a score of 300, which was considered the limit of our assay resolution. was used to assess cell viability. PBMCs were specifically stimulated for 20 h in vitro with different ASFV isolates at a MOI of 0.2. RPMI, and 10 µg/mL of phytohemaglutinin (PHA, Sigma-Aldrich) was used as the control of the technique. Any sample scoring ≥300 spots/500,000 PBMCs received a score of 300, which was considered the limit of our assay Viruses 2021, 13, 1678 resolution. 5 of 13 2.5.3. Virus Quantification 2.5.3.Titrating ASFV by hemadsorption is not always possible when working with non- Virus Quantification hemadsorbing Titrating ASFVstrains,bysuch as BA71∆CD2. hemadsorption is notAiming to adoptwhen always possible a reliable working comparative with non- method for ASFV titration of multiple samples, pig sera, collected hemadsorbing strains, such as BA71∆CD2. Aiming to adopt a reliable comparative at different timemethod points during the experiment, were used to quantify the ASFV virus DNA by for ASFV titration of multiple samples, pig sera, collected at different time points during real-time qPCR the [20]. Due to the fact that our qPCR technique had previously shown experiment, were used to quantify the ASFV virus DNA by real-time qPCR [20]. Due to the more reproducible results fact thatinour seraqPCRthantechnique in whole had blood, virus inshown previously sera and noreproducible more viremia wasresults measured, in serabeing than aware in wholethatblood, the amount virus in of sera virusand present in blood no viremia wasafter challenge measured, withaware being Georgia2007/1 would that the amount be of around 1 log higher virus present in blood inafter magnitude. challenge Briefly, the viral genomic with Georgia2007/1 wouldDNAbe was aroundobtained from 1 log higher 200 in magnitude. Briefly, the viral genomic DNA was obtained from 200 µL of sera usingand µL of sera using the Nucleospin Blood kit (Macherey-Nagel, Düren, Germany) the then used as Blood Nucleospin template to amplify an 85 bp-long kit (Macherey-Nagel, Düren, fragment Germany)from and the thenASFV used serine protein as template to kinase amplify gene an 85 (R298L). bp-longPCR amplifications fragment from the were ASFVperformed in duplicate serine protein kinase gene using the corre- (R298L). PCR sponding standards amplifications for absolute were performed in quantification. duplicate usingResults are expressed the corresponding as log10 standards forGEC per absolute ml of sera. The detection quantification. Results are limit of the technique expressed as log10 GECwas set peratmL 103ofGEC/mL. sera. The detection limit of the technique was set at 103 GEC/mL. 3. Results and Discussion 3. Results 3.1. andofDiscussion Replication Recombinant Viruses in Swine Macrophage Cultures 3.1. Replication of Recombinant Viruses in Swine Macrophage Cultures PAMs infected with either BA71∆CD2f and BA71∆CD2EP153R followed indistin- PAMs guishable infected kinetics with either of infection BA71∆CD2f (Figure and BA71∆CD2EP153R 2). As expected, more than 95% of followed the PAMs indistin- were guishable kinetics of infection (Figure 2). As expected, more than infected by 72 h (hpi) (Figure 2A), showing also similar maximum titers of ASFV-DNA 95% of the PAMs were in infected by 72 h (hpi) (Figure 2A), showing also similar maximum their supernatant (Figure 2B). Conversely, at the same multiplicity of infection (MOI), titers of ASFV-DNA in their supernatant BA71∆CD2DP96R (Figure showed a 2B). delay Conversely, at thekinetics, in the infection same multiplicity with only of infection half (MOI), of the PAMs BA71∆CD2DP96R showed a delay in the infection kinetics, with only half of the PAMs becoming infected at 72 hpi and showing a maximum titer one log below of those achieved becoming infected at 72 hpi and showing a maximum titer one log below of those achieved by BA71∆CD2f and BA71∆CD2EP153R. by BA71∆CD2f and BA71∆CD2EP153R. % positive cells Figure 2. Comparative infection kinetics of the three recombinant viruses, BA71∆CD2f, BA71∆CD2EP153R and BA71∆CD2DP96R, Figure in PAMs. 2. Comparative (A) Percentage infection kinetics ofofinfected cells recombinant the three analyzed by flow cytometry viruses, and (B) ASFV BA71∆CD2f, DNA amount found BA71∆CD2EP153R and in the supernatant of infected PAMs at different time points after infection. BA71∆CD2DP96R, in PAMs. (A) Percentage of infected cells analyzed by flow cytometry and (B) ASFV DNA amount found in the supernatant of infected PAMs at different time points after infection. 3.2. Comparative Protective Efficacy Induced by BA71∆CD2EP153R and BA71∆CD2DP96R Recombinant 3.2. Viruses Comparative Protective Efficacy Induced by BA71∆CD2EP153R and BA71∆CD2DP96R Recombinant AimingViruses to confirm if the deletion of DP96R or EP153R reduced the residual viral loadAiming previously observed to confirm after if the immunization deletion of DP96Rwith BA71∆CD2 or EP153R reduced[12], thethe amountviral residual of ASFV load DNA of each previously recombinant observed virus was followed after immunization directly after with BA71∆CD2 [12],inoculation the amountuntil the day of ASFV DNA of the Georgia2007/1 challenge (Figure 3). No clinical signs were observed following the immunization. The virus titers of BA71∆CD2f -immunized animals before challenge, as well as the protection afforded, yielded similar results to our previous studies [12], with the exception of pig number 66, euthanized at 6dpc (Figure 5), which responded poorly to the vaccine (Figures 7A and 8) and was Unfortunately, two out of the six pigs immunized with either BA71∆CD2EP153R or BA71∆CD2DP96R showed low albeit detectable copy numbers of virus genome in sera, confirming that these new prototypes do not improve the safety of BA71∆CD2. the protection afforded, yielded similar results to our previous studies [12], with the ex- ception of pig number 66, euthanized at 6dpc (Figure 5), which responded poorly to the vaccine (Figures 7A and 8) and was Unfortunately, two out of the six pigs immunized with either BA71∆CD2EP153R or BA71∆CD2DP96R showed low albeit detectable copy Viruses 2021, 13, 1678 numbers of virus genome in sera, confirming that these new prototypes do not improve 6 of 13 the safety of BA71∆CD2. Figure 3. Rectal temperature and ASFV DNA amount found in sera after immunization with BA71∆CD2f (A,B) BA71∆CD2EP153R (C,D) or BA71∆CD2DP96R Figure 3. Rectal temperature and ASFV DNA(E,F) in each amount individual found in seraanimal. Average and with after immunization standard deviation(A,B) BA71∆CD2f val- BA71∆CD2EP153R ues from control pigs(C,D) or BA71∆CD2DP96R immunized (E,F) inASFV with PBS are shown. each DNA individual amountsanimal. Average are plotted on and standard scale a logarithmic deviation values as GEC per 3 milliliter of serum, 10 GEC/mL being the limit of detection of the assay. Twenty-four days after immunization, all pigs were intramuscularly challenged with a lethal dose of 103 GEC of the highly pathogenic Georgia2007/1 isolate. Control pigs were euthanized by day 8 post-challenge, while only one out of the six pigs vaccinated with BA71∆CD2f (animal 66) died after Georgia2007/1 challenge (Figure 4). One and two out of the six pigs inoculated with BA71∆CD2DP96R and BA71∆CD2EP153R, respectively, did not survive the lethal ASFV challenge (Figure 4). Twenty-four days after immunization, all pigs were intramuscularly challenged with a lethal dose of 103 GEC of the highly pathogenic Georgia2007/1 isolate. Control pigs were euthanized by day 8 post-challenge, while only one out of the six pigs vaccinated with BA71∆CD2f (animal 66) died after Georgia2007/1 challenge (Figure Viruses 2021, 13, 1678 4). One and two out of the six pigs inoculated with BA71∆CD2DP96R and 7 of 13 BA71∆CD2EP153R, respectively, did not survive the lethal ASFV challenge (Figure 4). Figure 4. Survival rates after Georgia2007/1 challenge. Pigs were immunized with either BA71∆CD2f, BA71∆CD2DP96R Figure or BA71∆CD2EP153R 4. Survival rates and then after Georgia2007/1 challenged challenge. Pigs 24 daysimmunized were later with a with lethal either dose of Georgia 2007/1. BA71∆CD2f, BA71∆CD2DP96R or BA71∆CD2EP153R and then challenged 24 days later with a le- thal dose of Georgia 2007/1. Pigs that succumbed to Georgia2007/1 challenge (six controls and four immunized with recombinant Pigs that succumbed LAVs)toshowed clinicalchallenge Georgia2007/1 signs compatible withand (six controls acute fourASF (Figure 5), immunized including fever, loss of appetite, depression, apathy and a tendency with recombinant LAVs) showed clinical signs compatible with acute ASF (Figure 5), to huddling, as in- well as erythema and cyanosis. Conversely, surviving pigs did not show severe cluding fever, loss of appetite, depression, apathy and a tendency to huddling, as well as clinical signs after ASFV erythema and challenge (Figure 5). Three cyanosis. Conversely, out of surviving thedid pigs sixnot pigs immunized show with BA71∆CD2f severe clinical signs after remained ASFV clinically challenge (Figurenormal, while 5). Three outthe ofother the sixtwo showed pigs only a with immunized transient rise in rectal BA71∆CD2f re- temperature. mained clinicallyThus, pig number normal, while the63other showed two febricula showed only (~40 a◦ C) from day transient rise8in torectal 14 pc,tem- while pig number ◦ C). perature. Thus,62pighadnumber fever only at day 8febricula 63 showed post challenge (~40 °C)(rectal fromtemperature day 8 to 1441 ◦°C; orange) rectal temperature, while euthanized animals (green), mild (yellow) and high (>41 C; orange) rectal temperature, while euthanized animals are are indicated in red. Clinical scores are indicated with numbers; 1 being low-grade fever (<41◦°C) as the indicated in red. Clinical scores are indicated with numbers; 1 being low-grade fever (<41 C) as the only clinical observation. only clinical observation. Rectal temperature Rectal temperaturealmost almostperfectly perfectly matched matchedthe theamount amountof ofASFV ASFVDNA DNAdetected detectedin in serum (Figure 6) after challenge. All control pigs immunized serum (Figure 6) after challenge. All control pigs immunized with PBS were euthanized with PBS were euthanized byday by day6–86–8 after after the the lethal lethal challenge challengeshowing showingsignificant significantfeverfeverandandhigh highamounts amountsof ofASFV ASFV DNA in DNA insera. sera. As Asexpected, expected,surviving surviving pigspigsshowed showedsignificantly significantly lower loweramounts amountsof ofASFV ASFV DNA than control pigs (Figure 6). The massive release of immature DNA than control pigs (Figure 6). The massive release of immature and defective ASFV and defective ASFV particles from lysed infected macrophages at the last stage of the particles from lysed infected macrophages at the last stage of the ASFV infection, before ASFV infection, before thedeath the deathof ofthe theanimals, animals,explains explainsthe thenon-linear non-linearcorrelation correlationbetween betweenGECsGECs(10 (10 GEC/mL) 1010GEC/mL) andHAU and HAU(~10 (~10 HAU/mL) 88 HAU/mL) in in these these samples samples (not shown). Independently (not shown). Independently of of this, this, the thelevel level of virus of virusin insera seraofofsurviving survivingpigs pigsvaccinated vaccinatedwith withBA71∆CD2f BA71∆CD2f (Figure (Figure6A,B) 6A,B)was wasalways always 4–5 logarithms 4–5 logarithms belowbelow that thatfound foundin incontrol controlanimals. animals.Furthermore, Furthermore,ASFV ASFVpresence presencenevernever lastedmore lasted morethan thanoneoneweek, week,becoming becomingfree free ofof virus virus byby thethe time time of of sacrifice, sacrifice, with with pigpig num- number ber 65 not showing any detectable ASFV genome in its serum 65 not showing any detectable ASFV genome in its serum throughout the experiment. Thethroughout the experiment. The level level of virus of virus in serainfrom sera pigs fromimmunized pigs immunized with BA71∆CD2EP153R with either either BA71∆CD2EP153R (Figure(Figure 6C,D) 6C,D) or or BA71∆CD2DP96R BA71∆CD2DP96R (Figure(Figure 6E,F) was 6E,F) alsowas also remarkably remarkably decreased decreased compared compared to controlto control animals. animals. Nevertheless, Nevertheless, the reduction the reduction in virus in virus titers titersfrom in sera in sera from these these was animals animals was slightly slightly lower thanlower than that that found in thefound animals inimmunized the animals withimmunized with the the single mutant single mutant BA71∆CD2f, and several pigs showed BA71∆CD2f, detectable and several virus bydetectable pigs showed the end ofvirus the experiment. by the end of the experiment. Viruses 2021, 13, x FOR PEER REVIEW 9 of 13 Viruses 2021, 13, 1678 9 of 13 Rectal temperature (ºC) Rectal temperature (ºC) Rectal temperature (ºC) Figure 6. Rectal temperatures measured and ASFV DNA amount found in sera of immunized and control pigs after Geor- gia2007/1 challenge. Figure 6. Rectal Data plotted temperatures correspond measured to individual and ASFV animals DNA amount showing found rectal in sera temperatures of immunized (A,C,E) and and control virus pigs loads after in Geor- serum (B,D,F). gia2007/1 UpperData challenge. panels show correspond plotted data corresponding to BA71∆CD2f to individual immunized animals showing pigs, rectal middle panels temperatures to BA71∆CD2EP153R (A,C,E) and virus loads in serum and (B,D,F).to Upper lower panels panels show BA71∆CD2DP96R. data corresponding Average to BA71∆CD2f and standard deviation immunized values obtained frompigs, controlmiddle animalspanels to are also BA71∆CD2EP153R depicted. and Virus titers arelower panels plotted on a to BA71∆CD2DP96R. logarithmic Average scale as GEC and standard per milliliter deviation of serum, values obtained 103 GEC/mL fromthe serum being control limit animals of are also detection of thedepicted. Virus assay. * Day oftiters are plotted the death on a logarithmic of the animals. Last datascale as GEC recorded forper pigmilliliter of serum, 75 correspond to day GEC/mL 1036pc (deathserum date). being the limit of detection of the assay. * Day of the death of the animals. Last data recorded for pig 75 correspond to day 6pc (death date). 3.3. Evaluation of the Immune Response Induced by BA71∆CD2EP153R and BA71∆CD2DP96R 3.3. Evaluation of thekinetics The antibody Immuneobserved Responsebefore Inducedand by BA71∆CD2EP153R after challenge wereand as expected for both BA71∆CD2DP96R controls and BA71∆CD2f immunized pigs [12], with the only exception being pig number The antibody 66, which showedkinetics observed significant lower before ODs thanand the afterrest challenge were as expected of the animals within theforgroup both (Figure 7A). controls The fact thatimmunized and BA71∆CD2f this pig also showed pigs the lowest [12], with ASFV-specific the only exception beingT-cell pigresponse number (Figure 66, 8) may which showedsuggest the possibility significant of a human/mechanical lower ODs than the rest of the failure animalsduring withinvaccination, the group although (Figure a simple 7A). vaccine The fact failure that this cannot pig also be ruled showed the out. TheASFV-specific lowest severity of the clinical T-cell signs response observed after challenge (highest score of 12 recorded by day 6, forcing a humane (Figure 8) may suggest the possibility a human/mechanical failure during vaccination, endpoint) might be aexplained although by either simple vaccine an exacerbation failure cannot be ruled effect out.due Thetoseverity suboptimal of theimmunization clinical signs Viruses 2021, 13, x FOR PEER REVIEW 10 Viruses 2021, 13, 1678 10 of 13 observed after challenge (highest score of 12 recorded by day 6, forcing a humane point) might be explained by either an exacerbation effect due to suboptimal immu tion as has been described before [2,22] and/or some concomitant health affection. De as has been described before [2,22] the importance of theand/or ELISA some as an concomitant health affection. indicator of successful Despite there is immunization, the importancetotalof the ELISA as an indicator of successful immunization, there correlation between the level of ASFV-specific antibodies measured is not aby ELISA total correlationprotection. between the level of ASFV-specific antibodies measured Thus, pigs 74 and 75 immunized with BA71∆CD2EP153R andby ELISA andpig 81 im protection. Thus, pigs 74 and 75 immunized with BA71∆CD2EP153R and pig 81 immunized ized with BA71∆CD2DP96R died after challenge, despite showing similar antibody l with BA71∆CD2DP96R to survivingdied after pigs at challenge, the time of despite showing Georgia2007/1 similar antibody challenge. levels Conversely, pigsto80 and 82 surviving pigs at the time vived of Georgia2007/1 the lethal challenge. challenge, despite Conversely, showing slightly pigs lower80ODs. and 82 survived the lethal challenge, despite showing slightly lower ODs. Figure 7. ASFV-specific antibodies detected in serum of pigs immunized with BA71∆CD2f (A), BA71∆CD2EP153R Figure 7. ASFV-specific (B) and antibodies BA71∆CD2DP96R detected (C), in serum of pigs before andwith immunized after Georgia2007/1 BA71∆CD2f challenge. Indi- (B) and (A), BA71∆CD2EP153R vidual ELISA BA71∆CD2DP96R optical (C), before anddensity (OD) values challenge. after Georgia2007/1 are shownIndividual for each immunization ELISA optical group. density Average andare shown (OD) values standard deviation OD values obtained from the control group are also shown in each figure panel. The ELISPOT data (Figure 8) confirmed the successful immunization of most of the animals, albeit that again, no correlation was observed between the number of ASFV-spe- cific T cells present in blood and protection (confirming previous results). Thus, pigs 74, 75 and 81 were good responders and died after Georgia2007/1 challenge, while several Viruses 2021, 13, 1678 survivors showed low responses at the time of challenge. 11 of 13 Figure 8. IFNγ-secreting specific T cells found in peripheral blood mononuclear cells (PBMCs) in vitro. Figure 8. IFNγ-secreting specific≥T300 Any sample scoring spots/500,000 cells PBMCs received found in peripheral a score of 300, blood mononuclear which cells was consid- (PBMCs) in ered vitro. Anythe limit of sample our assay scoring ≥300resolution. spots/500,000 PBMCs received a score of 300, which was considered the limit of our assay resolution. The ELISPOT data (Figure 8) confirmed the successful immunization of most of the animals, albeit that In conclusion, again, no deletion correlation mutants of thewasattenuated observed between the number recombinant of ASFV- ASF virus, specific T cells present in blood and protection (confirming previous results). BA71ΔCD2, decrease vaccine efficacy and do not increase its safety. The fine equilibrium Thus, pigs 74, 75 and 81 were good responders and died after Georgia2007/1 challenge, existing between live attenuated ASFV vaccine replication, safety and efficacy requires a while several more survivors profound showed study low and responses at thekey might provide time of challenge. lessons for the future. In conclusion, deletion mutants of the attenuated recombinant ASF virus, BA71∆CD2, decrease Author vaccine efficacy Contributions: and do not F.R. coordinated and increase edited theitstext safety. The writing fine and equilibrium together existing with S.P.-P., between D.P.G., M.V.B.,live attenuated N.A., ASFVledvaccine J.A. and M.L.S. replication, the collaboration safetythe between and efficacy three requires institutions that a more par- ticipated in thestudy profound present work. and mightThis work forms provide part of for key lessons the the thesis work of E.L., finished last year future. under the coordination of S.P.-P. and F.R. at IRTA-CReSA, Barcelona. E.L. prepared the first version of the paperContributions: Author and the figuresF.R. herecoordinated presented. and F.A.,edited S.P.-P., theD.P.G. and M.V.B. text writing coordinated and together withthe exper- S.P.-P., D.P.G., imental work at IRTA-CReSA and USDA, respectively, and E.L., L.B.-C., J.Z., U.A., N.A., M.V.B., N.A., J.A. and M.L.S. led the collaboration between the three institutions that participated E.R.-M., E.V.,inM.M. and M.J.N. the present performed work. This workall forms the analytical work. part of the All authors thesis work ofcontributed E.L., finishedto the lastwriting and the year under revised the submitted version of the manuscript. All authors have read and agreed to the published coordination of S.P.-P. and F.R. at IRTA-CReSA, Barcelona. E.L. prepared the first version of the paper version andofthethe manuscript. figures here presented. F.A., S.P.-P., D.P.G. and M.V.B. coordinated the experimental work Funding: This work and at IRTA-CReSA was USDA, fundedrespectively, and E.L., by the Ministerio L.B.-C.,Innovación de Ciencia, J.Z., U.A., N.A., E.R.-M., E.V., y Universidades M.M. from theand Spanish Government (grant reference: AGL2016-78160-C2-1-R) with additional support from Hu- the M.J.N. performed all the analytical work. All authors contributed to the writing and revised submitted vepharma and version CERCAof the manuscript. from Generalitat deAllCatalunya authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement: Animal experiments were conducted according to animal ethics This Funding: welfare work wasapproved and protocols funded by bythe the Ministerio de Ciencia, Ethics Commission Innovación in Animal y Universidades Experimentation of thefrom the Spanish Generalitat de Government Catalunya (code(grantCEA-OH/9212/2, reference: AGL2016-78160-C2-1-R) in accordance to with additional (Directive the European support from Huvepharma 2010/63/EU) and CERCA and Spanish (Realfrom Generalitat Decreto 53/2013)deregulations Catalunya.under the supervision of the IRTA´s Ethical and Animal Institutional Welfare Review Committee. Board Statement: Animal experiments were conducted according to animal Datawelfare ethics Statement: Availability and protocols Theapproved by the Ethics data that support Commission the findings in Animal of this study Experimentation are available from the of the Generalitat corresponding authordeupon Catalunya (coderequest. reasonable CEA-OH/9212/2, in accordance to the European (Directive 2010/63/EU) and Spanish (Real Decreto 53/2013) regulations under the supervision of the IRTA’s Acknowledgments: Ethical and AnimalWe Welfare thank allCommittee. the personnel in charge of field studies and BSL3 facilities at IRTA- CReSA for their continuous help and Kevin Dalton for final editing of the manuscript. Finally, we Data thank Availability RLASB-ICTS andStatement: RISA from Thethedata that support Spanish the findings Government of thisAlliance and Global study are foravailable Researchfrom on the corresponding author upon reasonable request. Acknowledgments: We thank all the personnel in charge of field studies and BSL3 facilities at IRTA- CReSA for their continuous help and Kevin Dalton for final editing of the manuscript. Finally, we thank RLASB-ICTS and RISA from the Spanish Government and Global Alliance for Research on African swine fever (GARA) and World Organization for Animal Health (OIE) for their contribution. Conflicts of Interest: No conflicts of interest to declare. Viruses 2021, 13, 1678 12 of 13 References 1. Sánchez-Cordón, P.J.; Montoya, M.; Reis, A.L.; Dixon, L.K. African swine fever: A re-emerging viral disease threatening the global pig industry. Vet. J. 2018, 233, 41–48. [CrossRef] [PubMed] 2. Blome, S.; Gabriel, C.; Beer, M. Modern adjuvants do not enhance the efficacy of an inactivated African swine fever virus vaccine preparation. Vaccine 2014, 32, 3879–3882. [CrossRef] 3. Bosch-Camós, L.; López, E.; Rodriguez, F. African swine fever vaccines: A promising work still in progress. Porc. 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