rticle Feeding Spray-Dried Porcine Plasma to Pigs Improves the Protection Afforded by the African Swine Fever Virus (ASFV) Article BA71∆CD2 Vaccine Prototype against Experimental Challenge Feeding Spray-Dried Porcine Plasma to Pigs Improves the with the Pandemic Protection ASFV—Study Afforded by the African2 Swine Fever Virus (ASFV) BA71∆CD2 oan Pujols , Elena BlázquezVaccine 1,2,3 Prototype , Joaquim Segalés 1,4 , Fernandoagainst Rodríguez Experimental , Chia-Yu Chang , 2,3,5 Challenge 1,2,3 1,2,3 with the ordi Argilaguet Pandemic, Rosa , Laia Bosch-Camós 1,2,3 ASFV—Study 2 Rosell , Lola Pailler-García 1,2,3 , Boris Gavrilov , 2,6 1,2,3 7 oy Campbell and Javier Polo * 8 4,8, Joan Pujols 1,2,3 , Elena Blázquez 1,4 , Joaquim Segalés 2,3,5 , Fernando Rodríguez 1,2,3 , Chia-Yu Chang 1,2,3 , Jordi Argilaguet 1,2,31 , IRTA, Laia Centre Bosch-Camós 1,2,3 , Rosa Rosell 2,6 , Lola Pailler-García 1,2,3 , Boris Gavrilov 7 , de Recerca en Sanitat Animal (CReSA), 08193 Barcelona, Spain; joan.pujols@irta.cat (J.P.); Joy Campbell 8 4,8, andelena.blazquez@irta.cat Javier Polo * (E.B.); fernando.rodriguez@irta.cat (F.R.); chiayu.chang@irta.cat (C.-Y.C.); jordi.argilaguet@irta.cat (J.A.); laia.bosch@irta.cat (L.B.-C.); lola.pailler@irta.cat (L.P.-G.) 2 Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), 1 Campus de laIRTA, Universitat CentreAutònoma de Recercade enBarcelona (UAB),(CReSA), Sanitat Animal 08193 Barcelona, Spain; Spain 08193 Barcelona, 2 Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), joaquim.segales@irta.cat (J.S.); rosa.rosell@irta.cat (R.R.) Campus de 3 WOAH Collaborating la Universitat Centre Autònoma for Emerging de Barcelona and Re-Emerging Pig(UAB), 08193 Diseases Barcelona, in Europe, Spain IRTA-CReSA, 3 WOAH Collaborating Centre for Emerging and Re-Emerging Pig Diseases in Europe, IRTA-CReSA, 08193 Barcelona, Spain 4 APC Europe, 08193 Barcelona, S.L., 08403 Spain Spain Granollers, tation: Pujols, J.; Blázquez, E.; 4 5 Departament APC Europe, de Sanitat S.L., 08403 i Anatomia Granollers, Animals, Spain Facultat de Veterinària, Campus de la Universitat Autònoma galés, J.; Rodríguez, F.; 5 de Barcelona Departament (UAB), 08193 de Sanitat i Spain Barcelona, Anatomia Animals, Facultat de Veterinària, Campus de la Universitat Autònoma de hang, C.-Y.; Argilaguet, J.; 6 Departament Barcelona (UAB), 08193 d’Acció Climàtica, Barcelona, Alimentació Spain Rural, Generalitat de Catalunya, i Agenda osch-Camós, L.; Rosell, R.; 6 Departament 08007 Barcelona, Spain d’Acció Climàtica, Alimentació i Agenda Rural, Generalitat de Catalunya, iller-García, L.; Gavrilov, B.; et al. 08007 Barcelona, 7 Biologics Development, Spain 3A Nikolay Haytov Street, 1113 Sofia, Bulgaria; Huvepharma, 7 Biologics Development, Huvepharma, 3A Nikolay Haytov Street, 1113 Sofia, Bulgaria eding Spray-Dried Porcine Plasma boris.gavrilov@huvepharma.com 8 APC, LLC, 8 APC, LLC, Ankeny, IA joy.campbell@apcproteins.com 50021, USA Pigs Improves the Protection Ankeny, IA 50021, USA; fforded by the African Swine Fever * Correspondence: * Correspondence: javier.polo@apc-europe.com javier.polo@apc-europe.com rus (ASFV) BA71∆CD2 Vaccine Citation: ototype against Pujols, J.; Blázquez,Abstract: Experimental E.; This study This Abstract: aimed to evaluate study aimed the effects ofthe to evaluate feeding effectsspray-dried of feedingporcine plasma spray-dried (SDPP)plasma porcine on (SDPP) Segalés, hallenge with J.; Rodríguez, F.; Chang, the Pandemic the protection afforded on the by theafforded protection BA71∆CD2 African by the swine fever BA71∆CD2 virus African (ASFV) swine vaccine fever prototype. virus (ASFV) Two vaccine prototype. SFV–Study 2. Vaccines 2023, 11,J.;x.Bosch-Camós, C.-Y.; Argilaguet, groupsL.; of pigs acclimated to diets without or with 8% SDPP were intranasally inoculated with 105 Two groups of pigs acclimated to diets without or with 8% SDPP were intranasally inoculated with Rosell, R.; Pailler-García, L.; Gavrilov, ttps://doi.org/10.3390/xxxxx 105 plaque-forming plaque-forming units (PFU) of units live attenuated (PFU) of liveASFV strain BA71∆CD2 attenuated ASFV strain and, three weeks BA71∆CD2 later, and, threeleftweeks later, B.; et al. Feeding Spray-Dried Porcine cademic Editor: François Meurens in direct contact left in with direct pigs infected contact with with pigs the pandemic infected with Georgia the pandemic 2007/01 ASFV Georgia strain. 2007/01 During ASFV the strain. During Plasma to Pigs Improves the post-exposure (pe) period, 2/6(pe) the post-exposure from the conventional period, 2/6 from the diet group showed conventional dietagroup transient peakarectal showed tem- peak rectal transient Protection eceived: 25 January 2023Afforded by the African perature >40.5 °C before temperature day >40.5 ◦ Cpe, 20 and before some day tissue 20 pe, samples and some collected tissue at 20 samples d pe from collected 5/6 at were 20 d pePCR+ from 5/6 were Swine evised: 6 March Fever Virus (ASFV) 2023 BA71∆CD2 Vaccine Prototype for ASFV, albeit PCR+ showing for ASFV, Ct values albeit much showing Cthigher values than much Trojan higher pigs. than Interestingly, Trojan pigs. the SDPP Interestingly, group the SDPP group ccepted: 6 April 2023 against did not Experimental Challenge with show did fever, not neither show PCR+ fever, in neither blood PCR+ nor in rectal blood swab nor at rectalanyswabtimeat pe, any and time none pe, of and the postmor- none of the postmortem ublished: 10 April 2023 the Pandemic ASFV—Study tem 2. collected tissuetissue collected samples werewere samples PCR+ for ASFV. PCR+ Differential for ASFV. Differential serum serum cytokine cytokineprofiles profilesamong among groups at Vaccines 2023, 11, 825. https://groups at vaccination, vaccination,and andaahigher highernumber number of of ASFV-specific ASFV-specific IFNϒ-secreting IFN -secreting T T cells cells in in pigs pigs fed fed with with SDPP soon doi.org/10.3390/vaccines11040825SDPP soonafter afterthe theGeorgia Georgia 2007/01encounter, 2007/01 encounter, confirmed confirmed thethe relevance relevance of Th1-like of Th1-like responses responses in ASFin protection. opyright: © 2023 by the authors. Li- ASF protection. We We believe believe that that our our result result shows shows that that nutritional nutritional interventions interventions might might contribute contribute to to improving future nsee MDPI,Academic Basel, Editor: François Meurens Switzerland. his article is an open access article improvingASF future ASF vaccination vaccination strategies. strategies. Received: stributed under 25 January the terms and con-2023 tions of the Revised: Creative Commons 6 March 2023At- bution (CC BY) license (https://cre- Keywords:Keywords: African swine fever; African ASFV; swine spray-dried fever; porcine plasma; ASFV; spray-dried vaccine; porcine challenge; plasma; vaccine; challenge; nutritional Accepted: 6 April 2023 ivecommons.org/licenses/by/4.0/). nutritional intervention intervention Published: 10 April 2023 1. Introduction Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. African swine fever virus (ASFV) is a large, enveloped, double-stranded DNA virus. This article is an open access article As the only member of the Asfarviridae family [1], it can infect domestic pigs and wild distributed under the terms and boars of all ages causing African swine fever (ASF). ASF is a notifiable disease to the World conditions of the Creative Commons Organization for Animal Health (WOAH, formerly OIE) and is the number one threat to Attribution (CC BY) license (https:// the global swine industry and a major limitation to global trading [2]. Since 2007, a virulent accines 2023, 11, x. https://doi.org/10.3390/xxxxx creativecommons.org/licenses/by/ www.mdpi.com/journal/vaccines ASFV strain (genotype II) has emerged, causing mortality rates up to 100%, and has spread 4.0/). Vaccines 2023, 11, 825. https://doi.org/10.3390/vaccines11040825 https://www.mdpi.com/journal/vaccines Vaccines 2023, 11, 825 2 of 13 in Europe, China, South-East Asia, and more recently, in the Dominican Republic and Haiti, where millions of animals have succumbed to the disease [3]. Currently, there is no commercial vaccine available to fight the ASF pandemic at a global level. The lack of efficacy of inactivated vaccines and the poor protection afforded so far with recombinant vaccines based on ASFV-specific antigens left live attenuated viruses (LAVs) as the short–medium term choice to develop ASFV vaccines [4–6]. The recent launching of the first commercial vaccine against ASFV in Vietnam was based on a recombinant deletion mutant lacking the I177L gene from the Georgia 2007 ASFV isolate [7] and reflects the high expectation that this technology has opened in the field. Unfortu- nately, vaccination in some regions of Vietnam was suspended due to unexpected pig deaths (https://www.reuters.com/world/asia-pacific/vietnam-suspends-african-swine- fever-vaccine-after-pigdeaths-2022-08-24/, accessed on 24 January 2023). This was most probably due to defects in vaccination implementation, reopening biosafety concerns about the use of ASF LAVs in the field [8]. Together with the necessary implementation of standardized protocols for registration and approval of ASFV vaccines, we should continue investing efforts to better understand the mechanisms involved in protection against ASF, aiming to develop the safest and most efficient preventive and therapeutic strategies. Both antibodies [9] and CD8+ cells [10] play important roles in protection, together with an appropriate innate immune response [11]. Recent work performed, among others, in our own laboratory has confirmed the key importance of Th1-like responses and specific cytotoxic T lymphocytes (CTLs) in protecting against ASF, independently of working with subunit experimental vaccines [12,13] or with BA71∆CD2, a cross-protective recombinant live attenuated virus [14]. In parallel to the current efforts to develop efficient vaccines against ASF, we and others have also demonstrated that the pig’s immune status and/or their microbiota significantly influence the disease outcome [15–17]. In the present study, we aimed to evaluate the effects of feeding pigs with spray-dried plasma (SDP) on the protection afforded by the BA71∆CD2 vaccine prototype. SDP derived from porcine (SDPP) or bovine (SDBP) origin are dry functional ingredients that are extensively used in pig starter diets and consistently improve performance, feed efficiency, and animal survival, especially under stressful conditions such as pathogen challenge [18]. SDP contains a diverse mixture of many functional compounds such as immunoglobulins, albumin, growth factors, biologically active peptides, transferrin, amino acids, and other molecules that have biological activity independent of their nutritional value. Although the modes of action of SDP are not completely known, it has been shown to modulate the efficiency of the immune system [19]. Based on the demonstrated capability of SDPP feeding to accelerate the induction of specific Th1-like responses and to delay experimental ASFV transmission and disease progression [20] (back-to-back submitted manuscript), the objective of this study was to evaluate the effects of feeding SDPP on the protection afforded by the BA71∆CD2 vaccine prototype. 2. Materials and Methods 2.1. Clinical Monitoring The clinical state of the animals and the end-point criteria were evaluated by scoring the ASF-compatible clinical signs following a previously reported guide [21] with slight modifications. A score from 0 to 5 according to severity was applied as follows: 0: no clinical signs; 1: mild pyrexia (39.6–40.0 ◦ C); 2: mild pyrexia (39.6–40.0 ◦ C) and mild clinical signs (skin, digestive); 3: moderate pyrexia (40.0–40.5 ◦ C) and mild–moderate clinical signs (distal ear spots, mild limp, lying down, but remaining alert); 4: moderate–high pyrexia (40.5–41 ◦ C) and moderate clinical signs (remains dormant, only stands up when touched, hesitant step, subcutaneous bleeding <10%, diarrhea, mild tremors); and 5: pyrexia higher than 41 ◦ C and moderate–severe clinical signs (generalized subcutaneous bleeding, ataxia, spasticity, clouding, prostration, bloody diarrhea). signs (distal ear spots, mild limp, lying down, but remaining alert); 4: moderate–high py- rexia (40.5–41 °C) and moderate clinical signs (remains dormant, only stands up when touched, hesitant step, subcutaneous bleeding <10%, diarrhea, mild tremors); and 5: py- rexia higher than 41 °C and moderate–severe clinical signs (generalized subcutaneous Vaccines 2023, 11, 825 bleeding, ataxia, spasticity, clouding, prostration, bloody diarrhea). 3 of 13 2.2. Study Design The study was approved by the Committee of Ethics and Welfare “Comitè d’Experi- 2.2. Study Design mentació Animal de la Generalitat de Catalunya” with the protocol approval number The study was approved CEA-OH/11387/1. by the Committee For this study, of Ethics 24 4-week-old and Welfare Landrace “Comitè x Large Whited’Experimentació male pigs were Animal de la Generalitat de Catalunya” with the protocol approval number used. Pigs were randomly divided into two separate groups at the IRTA-Monells CEA-OH/11387/1. animal For this and facility study, 24 4-week-old acclimated Landrace to their assignedx Large White diet (see male Table pigs 1, as were used. described Pigs wereetran- by Blazquez al. domly divided into two separate groups at the IRTA-Monells animal facility and acclimated [20]). to their assigned diet (see Table 1, as described by Blazquez et al. [20]). Thus, sixteen animals were fed a conventional diet with 10.09% soy protein concen- Thus, sixteen animals were fed a conventional diet with 10.09% soy protein concentrate, trate, and eight pigs were initially fed a diet supplemented with 8% SDPP (AP920 pro- and eight pigs were initially fed a diet supplemented with 8% SDPP (AP920 produced duced by APC Europe S.L.U.-Granollers, Spain), replacing soy protein. Then, 14 days by APC Europe S.L.U.-Granollers, Spain), replacing soy protein. Then, 14 days later, later, animals were moved to the IRTA-CReSA BSL-3 animal facility and were distributed animals were moved to the IRTA-CReSA BSL-3 animal facility and were distributed in two in two separate rooms (Rooms 1 and 2) divided in half (left and right) with fences, main- separate rooms (Rooms 1 and 2) divided in half (left and right) with fences, maintaining taining the same diet (8 fed with SDPP-supplemented diet and 16 with the conventional the same diet (8 fed with SDPP-supplemented diet and 16 with the conventional diet) diet) during 10 additional acclimation days, as described in Figure 1. The rooms contain during 10 additional acclimation days, as described in Figure 1. The rooms contain slatted slatted floors, and the environmental conditions for both rooms were set at 22 ± 2 °C and floors, and the environmental conditions for both rooms were set at 22 ± 2 ◦ C and relative relative humidity of 60 ± 5%. The air renewal was established to be 12 times/h. The feed humidity of 60 ± 5%. The air renewal was established to be 12 times/h. The feed was was provided provided each each morning morning between between 7:309:30 7:30 and anda.m. 9:30 a.m. Figure 1. Figure 1. Schematic Schematic representation representationof ofthe thestudy studydesign. design.Abbreviations: BSL3 Abbreviations: BSL3= biosafety level = biosafety 3; NS level 3; = Nasal swab; RS= rectal swab; pv = post-vaccination; pe = post-exposure; d = day; PBMCs = periph- NS = Nasal swab; RS= rectal swab; pv = post-vaccination; pe = post-exposure; d = day; PBMCs = eral blood mononuclear cells. peripheral blood mononuclear cells. After acclimation, After acclimation, eight eight pigs pigs from from Room Room 11 (left (left pen), pen), fed fed the the conventional conventional diet, diet, and and eight pigs from Room 2 (left pen), fed the SDPP diet, were intranasally immunized eight pigs from Room 2 (left pen), fed the SDPP diet, were intranasally immunized (1 mL (1 mL per nostril) with a dose of 1055 PFU of BA71ΔCD2 ASFV. The rest of the pigs (eight), four per nostril) with a dose of 10 PFU of BA71∆CD2 ASFV. The rest of the pigs (eight), four locatedin located inthe theright righthalf halfofofRoom Room 1 and 1 and four four in in thethe right right halfhalf of Room of Room 2, continued 2, continued to betofed be the conventional diet. Therefore, the donor animals (Trojans) for both groups were fed the same control diet. Nineteen days after vaccination (d19pv), each of the non-immunized animals (two groups of four pigs fed with the conventional diet) was intramuscularly infected with 1 mL of a lethal dose (103 GECs/mL) of the pandemic Georgia 2007/1 ASFV strain [22]. Finally, two days later, the fences were removed, allowing the direct contact of vaccinated pigs with the “Trojan” pigs previously infected with Georgia 2007/01 (day 0 post-exposure, d0pe), with a final 1:2 ratio of Trojan to vaccinated pigs. This proportion was considered optimal for the transmission of the virus by direct contact [14]. All Trojan pigs had to be sacrificed Vaccines 2023, 11, 825 4 of 13 between 3 and 7 days post-infection. The study ended at d20pe, 41 days after initiating the vaccination (Figure 1). Throughout the experimental period, pigs were fed ad libitum and observed for clinical signs and rectal temperature daily. Blood samples (10 mL tubes with EDTA) and nasal and rectal swabs were taken at d0pv (before intranasal vaccination), d7pv, d14pv, and d21pv (d0pe, the first day the Trojan and vaccinated pigs were in contact), d4pe, d7pe, d14pe, and d20pe (d41pv). At necropsy, lesions were registered, and samples of spleen, tonsil, and gastro-hepatic, submaxillary, and retropharyngeal lymph nodes were frozen and kept at −75 ◦ C until use. Once thawed, all samples were simultaneously analyzed with real-time PCR (qPCR) to detect ASFV virus loads, as described below [23]. ELISPOT analysis was conducted using fresh peripheral blood mononuclear cells (PBMCs) from blood obtained with EDTA at d21pv (d0pe) and at d9pe and d20pe. 2.3. Laboratory Analyses DNA extraction was performed using an Indimag Pathogen Kit (Indical Biosciences, Leipzig, Germany). Viremia was determined with qPCR analysis using the primers de- scribed by Fernández–Pinero et al. [23] and the probe ASF-VP72P1 described in the current OIE ASF chapter (Terrestrial Manual OIE, Section 3.9, Chapter 3.9.1 African Swine Fever Virus pages 1–18) with the following modification in the thermoprofile made by the Spanish National Reference Laboratory for ASF: 10 min at 95 ◦ C, 5 cycles 1 min at 95 ◦ C + 30 s at 60 ◦ C, 40 cycles 10 min at 95 ◦ C + 30 s at 60 ◦ C with fluorescence acquisition in the FAM channel at the end of each PCR cycle. According to these amplification settings, results were considered positive when Ct ≤ 30, inconclusive when Ct was between 30 to 35, and negative when Ct > 35. The differential detection of BA71∆CD2 was performed in blood samples after ex- posure using a recently described probe-based SYBR Green qPCR (Applied Biosystems, Waltham, MA, USA, Path-ID qPCR Master Mix), targeting the LacI reporter gene, only present in the genome of the BA71∆CD2 vaccine virus [14]. The detection limit of this qPCR was 20 copies/reaction, with a Ct value of 34.93, standardized using serial dilutions of a plasmid encoding the LacI gene. Seroconversion was determined with ELISA (INgezim PPA COMPAC, INGENASA; Madrid, Spain). Nasal and rectal swabs were analyzed for the presence of the ASFV viral genome using the procedures previously mentioned. Once the nasal and rectal swabs arrived at the laboratory, the end of the swab was cut and placed in a tube with 1 mL of PBS. Tubes were stored at −75 ◦ C until DNA extraction and analysis with qPCR. For each tissue sample, 0.1 g of tissue was diluted 1:10 and homogenized using sterile PBS and TyssueLyser II (Qiagen, Hilden, Germany); DNA extraction and qPCR were performed as detailed above. Plasma samples obtained from pigs were stored at −75 ◦ C until use. Once thawed, lev- els of IFNα, IFNγ, IL-1β, IL-10, IL-12/IL-23p40, IL-4, IL-6, IL-8, and TNFα in plasma were quantified using the Luminex xMAP technology following the manufacturer’s instructions (ProcartaPlex Porcine Cytokine & Chemokine Panel 1; ThermoFisher Scientific, Waltham, MA, USA). The concentrations of each cytokine were calculated using xPONENT software (Luminex, Austin, TX, USA). Levels of TGFβ and IL-17α were quantified with ELISA (KingFisher Biotech, Saint Paul, MN, USA, [DIY0730S-003] and Invitrogen [CHC1683Kit], Waltham, MA, USA, respectively), following the manufacturer’s instructions. PBMCs were purified from EDTA blood samples by density–gradient centrifugation with Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA). To quantify by ELISPOT assay the number of IFNγ secreting cells, fresh PBMC were stimulated for 16 h with BA71∆CD2 (vaccine prototype) and/or Georgia 2007/01 (pandemic challenge virus) at a multiplic- ity of infection (MOI) of 0.2. Commercial mAbs (Porcine IFNγ P2G10 and biotin P2C11, BD Biosciences Pharmingen, San Diego, CA, USA) at 5 µg/mL were used, as previously described [24] (Díaz and Mateu 2005). Plates were revealed using HRP-conjugated Strepta- Vaccines 2023, 11, 825 5 of 13 vidin (Life Technologies, South San Francisco, CA, USA) and TMB substrate (MABTECH, Stockholm, Sweden), and spots were counted under a magnifying glass. 2.4. Statistical Analysis Data were analyzed as a completely randomized design using the GLM procedures of SAS (SAS Inst., Inc., Cary, NC, USA). An analysis of variance was conducted to detect differences among treatments. The independent variable was treatment. Dependent variables were body temperatures, blood, nasal and rectal swab, and tissue Ct values. The LSMEANS procedure was used to calculate the mean values by treatment. If treatment effects were detected, least squares means were separated using the PDIFF option in SAS. For cytokine statistical analysis, a linear mixed-effect model was constructed for each cytokine, with animal and replicate as fixed effects and treatment and day of study as random effects. In order to contrast both treatments at each time point, a post-hoc analysis was performed. The pig was considered the experimental unit. Means are considered significantly different if p < 0.05, while trends are reported as p = 0.05 to 0.10. 3. Results In the group fed the conventional diet, one animal died before starting the vaccination period. Therefore, this group started with 7 animals instead of 8. In addition, another animal (#396) died in this group on d31pv due to acute meningitis. In the SDPP group, 1 animal (#391) was euthanized on d21pv to balance both groups to 7 animals during the exposure period. This animal (#391) was randomly selected between animals within the corresponding pen, excluding those showing the lowest and the highest rectal temperature. Another animal (#376) died on d35pv due to intestinal prolapse. Both groups finished at d41pv with 6 pigs. All pigs intranasally vaccinated with 105 PFU of BA71∆CD2 survived the direct- contact challenge with pigs infected with Georgia 2007/01 independently of their assigned diet. However, differences were observed between treatment groups, including clinical Vaccines 2023, 11, xsigns FOR PEER andREVIEW viral load in blood, excretions, and tissues after exposure to the ASFV-infected 6 of 1 Trojan pigs. Interestingly, no fever was recorded at any time after Georgia 2007/01 challenge in any of the pigs from the SDPP group (Figure 2A; Supplementary Table S1). Figure 2. Rectal Figure 2. Rectal temperature temperature recorded individually recorded individually after2007/01 after Georgia Georgia 2007/01 exposure exposure of pigsofin- pigs intrana sally inoculated5 with 105PFU of BA71∆CD2. Data from pigs fed (A) the SDPP diet (SDPP1-7 pigs) o tranasally inoculated with 10 PFU of BA71∆CD2. Data from pigs fed (A) the SDPP diet (SDPP1-7 (B) the conventional diet (C1–C7 pigs). T1–T8 (dashed lines): Trojan pigs used for the direct contac pigs) or (B) the conventional dietcolor, challenge. In red (C1–C7 pigs).that animals T1–T8 died(dashed lines): before the Trojan end of pigs used for the direct the study. contact challenge. In red color, animals that died before the end of the study. A proportion of pigs vaccinated with 105 PFU intranasally and fed the conventiona diet (C3 and C4) showed a peak of fever starting at d14pe (Figure 2B), with no other sign of ASF infection observed. In addition to elevated rectal temperature, pigs #C3 and #C4 were the only ones ex hibiting significant virus loads in both their blood and nasal swabs at the end of the ex Figure 2. Rectal temperature recorded individually after Georgia 2007/01 exposure of pigs intrana sally inoculated with 105PFU of BA71∆CD2. Data from pigs fed (A) the SDPP diet (SDPP1-7 pigs) o Vaccines 2023, 11, 825 (B) the conventional diet (C1–C7 pigs). T1–T8 (dashed lines): Trojan pigs used for the 6 ofdirect 13 contac challenge. In red color, animals that died before the end of the study. A proportion of pigs vaccinated with 105 PFU intranasally and fed the conventiona A proportion of pigs vaccinated 5 diet (C3 and C4) showed with a peak10of PFU fever intranasally and (Figure starting at d14pe fed the 2B), conventional with no other sign diet (C3 and C4) showed a peak of of ASF infection observed. fever starting at d14pe (Figure 2B), with no other signs of ASF infection observed. In addition to elevated rectal temperature, pigs #C3 and #C4 were the only ones ex In addition to elevated hibiting rectal significant virus temperature, loads in bothpigstheir#C3 and blood #C4 and were nasal the at swabs only the ones end of the ex exhibiting significant virus loads in both their blood periment (Figure 3B; Supplementary Table S2). and nasal swabs at the end of the experiment (Figure 3B; Supplementary Table S2). Figure Figure 3. Ct values 3. Ct values obtained obtained by qPCR usingby qPCRnasal blood, usingand blood, nasal rectal andfrom swabs rectalindividual swabs from individual animals at animal different times at different times post-Georgia post-Georgia 2007/01 2007/01 exposure (pe). exposure Data from(pe). pigsData fed from pigs (A) the fed (A) SDPP dietthe SDPP diet (SDPP1 (SDPP1-7 pigs) or (B) the7conventional Vaccines 2023, 11, x FOR PEER REVIEW pigs) or (B) the conventional diet diet The (C1–C7 pigs). (C1–C7 pigs). higher theThe Cthigher values,the Ctlower the values,the theASFV lowerload, the ASFV load 7 orange of 14 show th with Ct values > 35 being considered negative (cut-off of the technique). Tables in with Ct values > 35 being considered negative (cut-off of the technique). Tables in orange show the average Ct values obtained in samples from Trojan pigs. Animals in red color died before the end average Ct values obtained of the in samples from Trojan pigs. Animals in red color died before the end of experiment. the experiment. Pigs fed the SDPP-containing diet did not exhibit fever or become viremic, and virus was notPigsdetected in rectal swabs atdiet fed the SDPP-containing anydidtime post-Georgia not exhibit fever2007/01 or becomeexposure (Figure viremic, 3A; and virus Supplementary Table S2). was not detected in rectal swabs at any time post-Georgia 2007/01 exposure (Figure 3A; In addition,Table Supplementary the vaccine S2). provided sterilizing protection in pigs fed the SDPP-contain- ing diet, as no virus In addition, thewas detected vaccine in tissue provided samples sterilizing at the end protection of the in pigs fedstudy (Figure 4A). In the SDPP-containing contrast, virus diet, as no waswas virus present in most detected of the samples in tissue pigs fromatthe theconventional diet group end of the study in at (Figure least 4A). In one organvirus contrast, at d20pe was (Figure present 4B; Supplementary in most Tablethe of the pigs from S3).conventional diet group in at least one organ at d20pe (Figure 4B; Supplementary Table S3). Figure Figure4.4.Ct Ctvalues valuesobtained obtainedby byqPCR qPCRfrom fromdifferent differenttissue tissuesamples samplesofofindividual individualanimals animalsat atthe theend end of of post-Georgia 2007/01 exposure (d20pe). Data from pigs (A) fed the SDPP diet (SDPP1-7 pigs)or post-Georgia 2007/01 exposure (d20pe). Data from pigs (A) fed the SDPP diet (SDPP1-7 pigs) or (B) (B)the the conventional conventionaldiet diet(C1–C7 (C1–C7pigs). pigs).The Thehigher higherthe theCtCtvalues, values,the thelower lowerthe theASFV ASFVload, load,with withCt Ct values > 35 being considered negative (cut-off of the technique). Tables in orange show the average values > 35 being considered negative (cut-off of the technique). Tables in orange show the average Ct values obtained in samples from Trojan pigs. Animals in red color died before the end of the Ct values obtained in samples from Trojan pigs. Animals in red color died before the end of the experiment. Abbreviations: Sub LN = Submaxillary lymph nodes; Retro LN = Retropharyngeal experiment. lymph nodes; Abbreviations: Sub LN = Submaxillary Gastro LN = Gastro-hepatic lymph nodes.lymph nodes; Retro LN = Retropharyngeal lymph nodes; Gastro LN = Gastro-hepatic lymph nodes. Without exception, the average Ct values found in vaccinated pigs, both in fluids during the exposure period and in postmortem tissues, were much higher than those found in Trojan pigs succumbing to the ASFV challenge (see bottom panels in Figures 3 and 4; Supplementary Table S2 and S3) confirming solid protection afforded by BA71∆CD2. Finally, in all cases, the only virus detectable after Georgia 2007/01 exposure Vaccines 2023, 11, 825 7 of 13 Without exception, the average Ct values found in vaccinated pigs, both in fluids during the exposure period and in postmortem tissues, were much higher than those found in Trojan pigs succumbing to the ASFV challenge (see bottom panels in Figures 3 and 4; Supplementary Tables S2 and S3) confirming solid protection afforded by BA71∆CD2. Finally, in all cases, the only virus detectable after Georgia 2007/01 exposure was the pan- demic virus, with no detectable traces of the BA71∆CD2 vaccine prototype (Supplementary Table S4). No treatment differences were observed (p > 0.1) in the kinetics of induction of ASFV- specific IgG and IgA in sera between either treatment group of pigs before and after Vaccines 2023, 11, x FOR PEER REVIEW 8 of 14 the challenge, except for d20pe (d40pv) in which the average values for IgG and IgA in conventional pigs was higher (p < 0.039 for IgG and p < 0.092 for IgA) (Figure 5A,B; Supplementary Tables S5 and S6). Figure Figure5.5.ASFV-specific ASFV-specificimmune immuneresponses responsesanalyzed analyzedininBA71∆CD2-vaccinated BA71∆CD2-vaccinatedpigs pigsbefore beforeand andafter after Georgia Georgia2007/01 2007/01challenge. ASFV-IgG challenge. ASFV-IgG (A)(A) and IgA and IgA(B)(B) antibodies from antibodies fromsera seraquantified quantified bybyELISA. ELISA. Average Averagevalues valuesfor forSDPP-fed SDPP-fedpigs pigs(SDPP1-7) (SDPP1-7)areareshown shownasasdashed dashedlines, lines,while whilesolid solidlines linesrepresent represent average average values for pigs fed the conventional diet (C1–C7). Standard deviation valuesshown. values for pigs fed the conventional diet (C1–C7). Standard deviation values are (C) are shown. Number of ASFV-specific IFNγ-secreting T cells found at d9pe by ELISPOT in PBMCs upon in vitro (C) Number of ASFV-specific IFNγ-secreting T cells found at d9pe by ELISPOT in PBMCs upon stimulation with either the pandemic Georgia 2007/01 (white boxes) or the BA71∆CD2 vaccine in vitro stimulation with either the pandemic Georgia 2007/01 (white boxes) or the BA71∆CD2 (black boxes). * = p < 0.05; # = p < 0.1. vaccine (black boxes). * = p < 0.05; # = p < 0.1. Similarly, treatment differences were not found (p > 0.1) between the number of ASFV Similarly, treatment differences were not found (p > 0.1) between the number of specific IFNγ-secreting T cells present at d0pe and d20pe (Supplementary Table S7). How- ASFV specific IFNγ-secreting T cells present at d0pe and d20pe (Supplementary Table S7). ever, by d9pe, the number of specific T cells secreting IFNγ upon ASFV stimulation was However, by d9pe, the number of specific T cells secreting IFNγ upon ASFV stimula- numerically higher for the vaccine virus (BA71∆CD2), and a trend to be higher (p = 0.07) tion was numerically higher for the vaccine virus (BA71∆CD2), and a trend to be higher for (p the Georgia = 0.07) 2007/01 for the Georgiapandemic 2007/01 ASFV strain pandemic in pigs ASFV fedin strain the SDPP pigs fed diet than the the SDPP dietconven- than the tional diet (Figure conventional diet5C). (Figure 5C). Finally, Finally,the theimmunomodulatory immunomodulatoryinfluence influenceofofthetheSDPP-containing SDPP-containingdietdietwas wasevident evident after after 24 days of feeding, even before starting the vaccination. Indeed, pigs fedwith 24 days of feeding, even before starting the vaccination. Indeed, pigs fed withSDPP SDPP showed showeda amodest modestbut butdetectable detectableamount amountininserum serumofofboth bothpro-inflammatory pro-inflammatory(IL-8(IL-8and and TNFα) TNFα)andandanti-inflammatory anti-inflammatorycytokines cytokines(IL-4) (IL-4)atatvaccination vaccinationtime time(Figure (Figure6). 6). Vaccines 2023, 11, x FOR PEER REVIEW 9 of 14 Vaccines 2023, 11, 825 8 of 13 Figure6.6.Serum Figure Serumconcentrations concentrationsofofseveral severalcytokines cytokinesquantified quantifiedby byLuminex Luminexafter after2424days daysofofdiet diet acclimation(before acclimation (beforeimmunization). immunization).* *= =p p<<0.05; 0.05;##==pp<< 0.1. 0.1. Interestingly, Interestingly,thethe levels levelsof the pro-inflammatory of the pro-inflammatory cytokine IL-17 IL-17 cytokine beforebefore vaccination were vaccination lower were in the SDPP lower in the group than inthan SDPP group pigsinfedpigswith fedawith conventional diet, and, a conventional diet, as expected, and, the as expected, levels of cytokines in serum varied along the experiment. Furthermore, the levels of cytokines in serum varied along the experiment. Furthermore, the Th1 cyto- the Th1 cytokine IL-12/IL-23p40 increased kine IL-12/IL-23p40 in the increased in SDPP the SDPPgroup groupat 7atdpv 7 dpv(Figure (Figure6),6), coinciding coincidingwithwiththe the elevated elevated levels of ASFV-specific T cells observed with ELISPOT (Figure 5C). Levelsofofthe levels of ASFV-specific T cells observed with ELISPOT (Figure 5C). Levels the two twocytokines cytokinesIFNγ IFNγand andTNFα, TNFα,also alsorecently recentlyidentified identifiedasasmarkers markersofofvaccine-induced vaccine-induced ASFV-specific ASFV-specificTh1 Th1response response[14],[14],showed showeda apeakpeakatat4 4dpe dpesignificantly significantlyhigher higherininanimals animals fed with SDPP. Despite TGFβ seeming to show some significant differences, fed with SDPP. Despite TGFβ seeming to show some significant differences, they mostly they mostly corresponded correspondedtotothe theoutlier outliervalue valueobserved observedfor forone onepigpigininthe theconventional conventionalgroup group(#C4), (#C4), coinciding with an animal showing fever and low Ct values. Finally, coinciding with an animal showing fever and low Ct values. Finally, IL-8, TNFα, IL-8, TNFα, andand IL-10IL- peaked at the 10 peaked endend at the of the study of the studyin the group in the groupof of pigs fedfed pigs thetheconventional conventional diet, diet,coinciding coinciding with withthe thedetection detectionofofASFV ASFVreplication replicationand andfever fever(Supplementary (SupplementaryTable TableS8). S8). 4. Discussion 4. Discussion Due to the wide expansion of ASFV around the world, leading to a negative impact Due to the wide expansion of ASFV around the world, leading to a negative impact on pig health as well as huge economic consequences for pig producers, research on new on pig treatmentshealth andasvaccines well as huge economic that could helpconsequences to mitigate the fornegative pig producers, researchrelated repercussions on new to this virus has intensified in recent years. Because inactivated and subunit vaccineto treatments and vaccines that could help to mitigate the negative repercussions related this virus has formulations intensified have failed toinprotect recentpigs years. Because against theinactivated pandemic ASFV,and subunit researchvaccine effortsformu- have lations have failed to protect pigs against the pandemic ASFV, focused on either natural or recombinant LAVs as the only short–medium-term strategy research efforts have tofo- cusedhighly obtain on either natural efficient ASForvaccines recombinant [4,25].LAVs as the Several only short–medium-term recombinant LAV candidates havestrategy beento obtain highly efficient ASF vaccines [4,25]. Several recombinant LAV candidates described so far in the literature, capable of inducing solid protection against experimental have been described so far in the literature, capable of inducing challenges with the pandemic genotype II ASFV strains [5,6]. solid protection against experimental challenges with the The detection of pandemic genotype II illegally introduced ASFV strains genotypes I and[5,6]. II attenuated ASFV vaccines in Chinese pig farms [2,26] confirms the need for cautionI when The detection of illegally introduced genotypes and II delivering attenuatedASFASFV vaccines LAVs to thein Chinese field pig the without farms [2,26] confirms appropriate the need supervision fromforregulatory caution when delivering agencies. This ASF led toLAVs to the renewed field without efforts the appropriate to standardize protocols supervision from regulatory for the registration and approvalagencies. of theThis mostled to renewed efficient and safest vaccines [27]. At the same time, it is important to continue searching for methodsand efforts to standardize protocols for the registration and approval of the most efficient to improve LAV prototypes and to develop efficient subunit vaccines for the future. Vaccines 2023, 11, 825 9 of 13 In the present experiment, we extended previous work using BA71∆CD2, a recombi- nant vaccine prototype capable of protecting in a dose-dependent manner against experi- mental challenges with homologous and heterologous viruses, including the genotype II pandemic virus [28,29]. For comparative studies, in the present work, an intranasal dose of 105 PFU of BA71∆CD2, one logarithm below the optimal dose previously reported [14], was administered to pigs fed a conventional diet with or without SDPP. As expected for the dose and route used, all pigs survived exposure to the Trojan pigs infected with Georgia 2007/01, independently of the dietary treatment. However, significant differences were observed between treatment groups. Tissues and fecal samples from all pigs consuming the SDPP-containing diet were negative for ASFV genome detection (at levels below our detec- tion methods) at all sampling times following exposure to the Trojan pigs. In contrast with this apparent sterilizing protection, many pigs fed the conventional diet showed detectable virus in one or more samples post-exposure to the Trojan pigs. It is important to notice that the Georgia 2007/01 virus titers found in fluids and tissues of some vaccinated pigs fed the conventional diet were higher than expected, at least compared with those previously found using lower and higher vaccine doses than the one tested here [14]. We believe that this might be due to a sub-optimal health status of the animals from origin, which might negatively affect ASFV vaccination and transmission, as has been postulated in [14,17]. In fact, and as described in the results section, we had three ASF-unrelated deaths, one of them even before starting the vaccination despite treating them with antibiotics. Independently of this reality, the virus titers found in vaccinated pigs were always below those found in the Trojan pigs succumbing to Georgia 2007/01, confirming the solid protection afforded by the vaccine, even in adverse conditions. Therefore, the current data suggest that dietary SDPP improved vaccine efficiency and demonstrates that dietary SDPP could have a direct benefit for ASF vaccination. Treatment differences, except at the end of the study, were not observed in the serum levels of anti-ASFV IgG and IgA antibodies. Further studies are needed to better understand the kinetics of induced immunoglobulin isotypes as well as the local immune responses induced at the site of immunization and ASFV entry (nasal mucosa) and other mucosal tissues to distinguish any changes in the antibody induction due to feeding diets with SDPP [30,31]. Conversely to the antibody kinetics, an increase in specific IFNγ secretory T cells was observed in vaccinated pigs, detectable by d9pe to the Trojan pigs. The fact that virus- specific T cells recognize both the BA71∆CD2 vaccine and the pandemic virus confirms the induction of T cells capable of recognizing genotypes I and II, currently circulating in China [2]. These results and the corresponding peak of serum TNFα early after Georgia 2007/01 exposure of SDPP-fed pigs is consistent with the relevance of vaccine-induced IFNγ + TNFα+ polyfunctional memory Th1 cells in ASFV protection [14] and in the delayed transmission of ASFV in SDPP-fed pigs [20]. Additionally, in this line, the peak of the Th1 cytokine IL12 [32] in plasma at 7 dpv also suggests the SDPP-driven enhancement of the vaccine-induced Th1 response. Altogether, these results indicate that the addition of SDPP to diet somehow enhances the vaccine-induced ASFV-specific cellular responses. Further studies focused on mucosal immunity and its interplay with systemic immune responses will be required to better characterize the mechanisms behind this observation. Of particular interest are the differences observed for several immune mediators in the serum of vaccinated pigs consuming the SDPP diet compared to that of pigs fed the conventional diet, even before immunization. Feeding SDPP resulted in elevated levels of pro- and anti-inflammatory cytokines in their plasma, confirming the tight regulation of the immune responses previously reported for SDPP-fed animals [33,34]. These results also confirm the relevance of the health and immune status of the animals in the responses observed after ASFV vaccination [15–17] and open new avenues for dietary intervention. Interestingly, the sequential increase in IL-17 (from d0pv to d21pv) in vaccinated pigs fed the conventional diet may indicate a tighter control of Th17 and T-regulatory cells favoring the optimal expansion of Th1-like responses [35]. In this regard, a recently Vaccines 2023, 11, 825 10 of 13 published study associated the presence of regulatory T cells with the lack of long-term memory responses induced by ASF LAVs [36]. Dietary SDPP was shown to improve vaccine efficiency in a commercial trial where pigs were vaccinated with a commercial vaccination program [37]. Finally, the increase in IL-10, IL-8, and TNFα at the end of the study in pigs fed the conventional diet is consistent with the presence of Georgia 2007/01 at this time point in the animals from this group. Of course, the theoretical dysregulation of these cytokines is much lower than previously described at a late time post-infection in naïve pigs showing acute ASF with an uncontrolled cytokine storm due to the massive ASFV presence [38–41]. SDPP is a functional feed ingredient that has been demonstrated to systemically mod- ulate the immune system. In some studies, the effects exerted by SDPP were characterized to promote both Th1-like response and cytokine induction. Díaz et al. [42] reported a signif- icant reduction in interstitial pneumonia and faster virus clearance in pigs infected with porcine reproductive and respiratory syndrome virus and consuming an SDPP-containing diet. The authors concluded that pigs fed SDPP were able to mount a more robust im- mune response and were able to clear the virus more efficiently. The increase in cytokine expression (IFNγ and IL-1) in the lungs of pigs receiving SDPP in their feed points to a Th1 enhancement. Markowska-Daniel and Pejsak [43] reported that the administration of SDPP through both water and feed significantly increased, especially in smaller pigs, the percentage of CD8+ T cells, which are critical for protection against ASFV [10]. Thus, dietary SDPP may enhance the immune response of pigs vaccinated with BA71∆CD2, contributing to sterilizing immunity observed in the present experiment. 5. Conclusions In summary, under the conditions of this exploratory study to prove the concept, the addition of SDPP in feed improved the ASFV vaccine prototype efficacy. Pigs fed the diet with SDPP showed lower virus load in nasal secretion and absence of virus in blood and feces after exposure to Trojan pigs infected with Georgia 2007/01 compared to those fed the conventional diet. Furthermore, no virus was detected in any organ tissue of the pigs fed the SDPP diet at the time of sacrifice (d20pe). This suggests that dietary SDPP can be used strategically as a health management tool to enhance vaccine efficiency. However, it is important to point out that the current work involved a limited number of animals under controlled conditions. These results should be verified and extended in further studies with a larger number of animals under field conditions. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/vaccines11040825/s1. Table S1: Rectal temperature throughout the duration of the study; Table S2: Individual PCR results (Ct values) by day in blood, nasal and rectal swab samples; Table S3: Individual PCR results (Ct values) on different tissues at the time of sacrifice; Table S4: Pigs vaccinated with BA71∆CD2 did not show detectable vaccine virus in blood after Georgia 2007/01 challenge; Table S5: individual IgG values (OD) on serum by day; Table S6: Individual IgA values (OD) on serum by day; Table S7: Elispot results at different days during the study; Table S8: Individual cytokines results at different days during the study. Author Contributions: Conceptualization, B.G., E.B., F.R., J.P. (Javier Polo), J.P. (Joan Pujols) and J.S.; methodology, C.-Y.C., E.B., F.R., J.A., J.P. (Joan Pujols), J.S., L.B.-C. and R.R.; software, F.R., L.P.-G., J.C., J.P. (Javier Polo) and J.P. (Joan Pujols); validation, F.R., J.A., J.P. (Javier Polo), J.P. (Joan Pujols) and J.S.; formal analysis, F.R., J.A., L.P.-G., J.C., J.P. (Joan Pujols), J.P. (Javier Polo) and L.B.-C.; investigation, C.-Y.C., F.R., J.A., J.P. (Joan Pujols), J.S. and L.B.-C.; resources, J.P. (Joan Pujols) and F.R.; data curation, E.B., F.R., J.P. (Javier Polo) and J.P. (Joan Pujols); original draft preparation, E.B., F.R., J.A., J.P. (Javier Polo), J.P. (Joan Pujols), J.S. and L.B.-C.; writing—review and editing, F.R., J.A., J.P. (Javier Polo), J.S. and L.B.-C.; visualization, F.R. and J.P. (Javier Polo); supervision, F.R. and J.P. (Javier Polo); project administration, J.P. (Joan Pujols); funding acquisition, J.P. (Javier Polo). All authors have read and agreed to the published version of the manuscript. Vaccines 2023, 11, 825 11 of 13 Funding: Funding for this study was provided by APC Europe, S.L.U., Granollers, Spain and APC LLc, Ankeny, US. Both companies manufacture animal blood products for animal consumption. The companies provided support in the form of salaries for authors E.B., J.C., and J.P. (Javier Polo) retrospectively. However, none of these companies had any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. We also acknowledge the support of the Spanish Ministry of Science and Innovation (grant reference PID2019-107616RB-I00). Institutional Review Board Statement: The study was approved by the committee of ethics and welfare “Comitè d’Experimentació Animal de la Generalitat de Catalunya” with the protocol approval number CEA-OH/11387/1. Informed Consent Statement: Not applicable. Data Availability Statement: All data from this study are provided in the manuscript and Supple- mentary Tables. Acknowledgments: We thank the technicians and Ph.D. students of IRTA-CReSA for helping in the experimental setup. Conflicts of Interest: The authors have read the journal’s policy, and the authors of this manuscript have the following competing interests: E.B. and J.P. (Javier Polo) are employed by APC Europe, S.L.U. Granollers, Spain; J.C. and J.P. (Javier Polo) are employed by APC LLC, Ankney, US. Both companies manufacture and sell spray-dried animal plasma; B.G. is employed by Huvepharma, Sofia, Bulgaria, a company that develops and commercializes vaccines for animal health. However, the companies had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. C.-Y.C., F.R., J.A., L.B.-C., J.P. (Joan Pujols), J.S., R.R., and L.P. declare no conflict of interest. This does not alter the authors’ adherence to all journal policies on sharing data and materials. References 1. Alonso, C.; Borca, M.; Dixon, L.; Revilla, Y.; Rodriguez, F.; Escribano, J.M.; ICTV Report Consortium. 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Markowska-Daniel, I.; Pejsak, Z. Immunological and production parameters in pigs fed spray-dried animal plasma. Bull. Vet. Inst. Pulawy. 2006, 50, 455–459. Available online: https://www.semanticscholar.org/paper/IMMUNOLOGICAL-AND- PRODUCTION-PARAMETERS-IN-PIGS-FED-Markowska-Daniel-Pejsak/56d66722cd8bb6148a42cdafbc1544b2793f241d (ac- cessed on 24 January 2023). Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.