Bosch-Camós et al. Porcine Health Management (2020) 6:17 https://doi.org/10.1186/s40813-020-00154-2 REVIEW Open Access African swine fever vaccines: a promising work still in progress Laia Bosch-Camós, Elisabeth López and Fernando Rodriguez* Abstract: African swine fever (ASF), a disease of obligatory declaration to the World Organization for Animal Health (OIE), has contributed to poverty and underdevelopment of affected areas. The presence of ASF has been historically neglected in Africa, contributing to its uncontrolled expansion and favouring its spread to continental Europe on at least three occasions, the last one in 2007 through the Republic of Georgia. Since then, African swine fever virus (ASFV) has spread to neighbouring countries, reaching the European Union in 2014, China in the summer of 2018 and spreading through Southeast Asia becoming a global problem. Lack of available vaccines against ASF makes its control even more difficult, representing today the number one threat for the swine industry worldwide and negatively affecting the global commerce equilibrium. Main body: In this review, we intend to put in perspective the reality of ASF vaccination today, taking into account that investment into ASF vaccine development has been traditionally unattractive, overall since ASF-free areas with large swine industries applied a non-vaccination policy for diseases listed by the OIE. The dramatic situation suffered in Asia and the increasing threat that ASF represents for wealthy countries with large swine industries, has dramatically changed the perspective that both private and public bodies have about ASF vaccinology, although this is controversial. The feasibility of modifying the ASFV genome has led to safe and efficacious experimental recombinant live attenuated viruses (LAVs). The main challenge today will be confirming the safety and efficacy of these technologies in the field, accelerating transfer to the industry for official registration and commercialization. The complexity of ASFV, together with the lack of knowledge about the mechanisms involved in protection and the specific antigens involved in it, requires further investment in research and development. Although far from the efficacy achieved by LAVs, subunit vaccines are the optimal choice for the future. If the world can wait for them or not is a contentious issue. Conclusion: Despite their inherent disadvantages, LAVs will be the first technology to reach the market, while subunit vaccines will need much further research to become a successful commercial reality. Keywords: African swine fever (ASF), African swine fever virus (ASFV), Live attenuated virus (LAV), Subunit vaccines, Antibodies, Cytotoxic CD8+ T-cells (CTLs), Protection, Biosafety Background 170–190 kb DNA genome, encoding 151–167 open African swine fever (ASF) is a swine hemorrhagic viral reading frames (ORFs) [1]. The ASFV virion is a com- disease that is of mandatory declaration to the World plex multi-enveloped and multi icosahedral particle con- Organization for Animal Health (OIE). The disease is taining at least 68 different structural viral polypeptides caused by African swine fever virus (ASFV), a large and 21 cellular proteins and possess a much more com- nucleocytoplasmic virus that contains a double-stranded plex structure than previously believed [1–3]. ASFV rep- licates predominantly in mononuclear-phagocytic cells * Correspondence: fernando.rodriguez@irta.cat (monocytes and macrophages), which play a front-line IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la role in activating and orchestrating the innate and Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bosch-Camós et al. Porcine Health Management (2020) 6:17 Page 2 of 14 adaptive host responses. During infection, ASFV express EU countries in 2014. In August–September 2018, ASFV multiple proteins that subverts several host pathways in was declared in China, the country that raises half of the order to ensure efficient virus replication in vivo [4]. world’s pig population and a year and a half later the ASF was described for the first time in Kenya in 1921 as situation can be described as a global epidemic threating a lethal hemorrhagic disease affecting the first European global swine production [11]. imported domestic pigs and being maintained asymp- tomatically in a sylvatic cycle between ticks of the genus The need of a vaccine for ASFV Ornithodoros and warthogs (Phacochoerus spp.), acting Lack of vaccines against ASF leaves the control of the as long-term carriers and reservoirs of ASFV [5]. The disease to an efficient and rapid diagnosis and the cul- complex live cycle of ASFV in this region explains the ling of infected and exposed animals (www.oie.int). This actual presence of more than 24 different ASFV geno- strategy has been demonstrated as inefficient in coun- types, classification based on the sequence of B646L, en- tries with limited resources, incapable of implementing coding p72, the major capsid protein [6]. Outside Africa, fair policies of compensation and ASF control. only genotypes I and II have been found and both do- Lack of efficient treatments or vaccines against ASF com- mestic pigs and wild boars (both being Sus scrofa), are plicate ASF control even more. Development of vaccines equally susceptible to the disease [7]. When introduced against ASF has been historically unattractive for the indus- into disease-free regions or domestic pig populations, try. On one hand, ASF remained endemic almost exclu- the disease predominantly shows acute forms with high sively in Africa, and on the other hand, ASF-free countries mortality rates up to 100%. After several years of ASFV with developed swine industries apply a non-vaccination presence in endemic areas, mortality rates decline due to policy for diseases of obligatory declaration to the OIE virus adaptation to the hosts and infected individuals (www.oie.int). Therefore, there is still a lot of work to per- show subacute forms of the disease or even no clinical form to provide safe and efficient vaccines and to guarantee signs, complicating even more its detection and eradica- their optimal application in the field [12]. However, the en- tion, as described in the Iberian Peninsula [8]. ASFV can trance of ASFV in China has totally changed the perception spread into neighboring areas carried by free-ranging in- of ASF vaccinology [13, 14]. Limited resources together fected wild animals but it can also emerge into new dis- with the complexity of the virus have contributed to tant areas by the illegal transport of infected pigs, the explaining the gaps existing today regarding ASFV infection use of contaminated pork or pork products for feeding and immunity. So far, the precise nature of the host pro- pigs, or even fomites such as equipment (boots, trucks, tective responses has not been fully determined and pro- etc.) not properly disinfected. Once established, the virus tective antigens remain to be identified, hampering the disseminates via oral-nasal routes by direct contact be- rational design of vaccines. Despite these limitations, the tween infected and non-infected individuals or with con- few laboratories devoted to work on ASF vaccinology, have taminated products [9]. Unfortunately, ASFV is one of contributed to several seminal findings in the field [15]. the most resistant viruses to chemical and physical in- Thus, we can confirm today: first, that vaccines against ASF activation, therefore complicating the tasks of disinfec- are not a utopia; second, that LAVs can be a short-medium tion. Transcontinental spread of ASFV from Africa first term reality to fight the disease, overall in endemic regions occurred in two consecutive waves: 1957 and 1960, or regions in danger to become endemic; and third, that when genotype I viruses emerged in Spain and Portugal, subunit vaccines can be a long-term vaccine choice. where it became endemic and spreading to other Euro- Collaborative efforts between academia and industry pean countries, South America and the Caribbean. The should allow the improvement of safety and DIVA properties eradication campaign from the European Continent (capable to Differentiate Infected from Vaccinated Animals) lasted more than 30 years and provoked enormous eco- of the experimental vaccine prototypes below described. This nomic losses in affected countries [8]. The complex should be the beginning of a new era for ASF vaccinology ASFV epidemiological situation above described in Af- where continuous collaborative research will be needed to rica, together with the absolute lack of intervention from obtain the safest and most efficient vaccine in the future. the rest of the world, facilitated the exportation of the Controlling the virus in Asia and Europe is mandatory for virus again to Europe, catching us totally off guard and the swine industry, but controlling ASFV in Africa should be unprepared. Thus, barely 10 years after declaring the mandatory for humanity. Even if we do not appeal to hu- continental European Union (EU) free of ASF, a second manitarian reasons, we should turn to common sense, since transcontinental outbreak occurred in 2007, when geno- reducing the epidemiological pressure of ASFV in Africa type II ASFV from Eastern Africa was first detected in should diminish the risk of exportation of new ASFV strains Georgia [10]. From the Caucasus, and with the involve- in the future. Collaboration between researchers in the field, ment of wild boars in the spread, ASFV broke first into private companies, governmental bodies and international Eastern Europe and Baltic Countries, reaching the first organisms such as the OIE and the Food and Agriculture Bosch-Camós et al. Porcine Health Management (2020) 6:17 Page 3 of 14 Organization of the United Nations (FAO) is necessary if we presence of hemagglutination inhibitory antibodies, the are willing to succeed in controlling ASF, today the number ability of these antibodies to in vitro inhibit ASFV infec- one global threat for animal health. tion and the capability of these animals to survive the ex- perimental challenge with the homologous virulent virus Classical vaccines: understanding the mechanisms involved [30]. in protection In our vision, both an adequate innate immune re- Classical vaccines based on inactivated ASFV, independ- sponse [31] together with adaptive responses, including ently of the inactivation method and the adjuvant used, antibodies and CD8+ T cells, will be essential to induce have proven ineffective [16, 17]. Despite inactivated vac- sterilizing immunity against ASFV. cines being very efficient at inducing antibodies, on oc- Despite their enormous efficacy, the use of ASF LAVs as casions capable of blocking the virus in fluids, they are vaccines in the field is still controversial [12], mainly due not very efficient at inducing specific cytotoxic CD8+ T- to biosafety concerns related to their inherent infectious cells (CTLs) [18], crucial for elimination of virus- nature. Natural attenuation of ASFV in endemic areas has infected cells. Incorporation of new adjuvant formula- complicated its eradication, mainly due to the difficult tions and novel and more innocuous inactivation proce- identification of chronically infected pigs that might act as dures, might contribute to designing efficient inactivated continuous reservoirs [8]. ASFV chronicity paralleled with vaccines in the future [1–3]. a total change of clinical findings [32]. The concomitant LAVs, whether naturally attenuated or adapted to tis- presence of virus and antibodies correlated with the pres- sue culture, have been used as experimental models to ence of lesions compatible with immune complex depos- understand the mechanisms involved in homologous ition, including necrotic foci in joint swelling [33–35]. protection against ASF. Antibody exacerbation phenomenon has been also ob- The presence of ASFV-specific CTLs was initially dem- served after immunization with inactivated virus and even onstrated using PBMCs from animals surviving the infec- after experimental immunization with subunit vaccines tion with attenuated viruses [19–21]. More recently, the [17, 36–38]. The fine balance existing between the pro- CTL activity has been associated to both CD8+ and double tective and detrimental role of ASFV-specific antibody re- positive CD4+/CD8+ T-lymphocytes, the latter thought to sponse needs further investigation, both from the basic include memory/effector T cells [22]. The definitive evi- and applied point of view. Understanding the intrinsic dence identifying ASFV-specific T-cells as crucial for mechanisms explaining the protection and the exacerba- ASFV protection came after the demonstration that tion caused by ASFV-specific antibodies is crucial for fu- in vivo depletion of CD8+ cells abrogated protective im- ture vaccine designs. Albeit not frequently reported, munity to ASFV [23]. Today we know that two distinct natural attenuation of ASFV has been also reported more phenotypes of porcine CTLs are capable to lyse ASFV- recently in the Caucasian region, mostly associated to wild infected cells: a conventional CD8 single positive, and a boars [39, 40]. Experimental infection with some of these CD4+CD8αβ+ phenotype, both being perforin positive and new isolates ratified their attenuation in domestic pigs showing swine leukocyte antigen I (SLA I)-restricted cyto- [40] and in wild boars [41]. Together with the polemic use toxicity. Increasing levels of circulating conventional of these ASFV strains as vaccines or as vaccine templates CD8+ T-lymphocytes have been correlated with the onset in the future [12, 41], their presence in nature open new of ASF clinical signs, while CD4/CD8αβ double positive concerns about the eradication measures today in place, CTLs were significantly increased in protected pigs [24]. based on detecting ASFV nucleic acid. The presence of at- Similarly, passive administration of antibodies or colos- tenuated ASFV isolates in the field deserves deeper studies trum from ASF convalescent pigs, conferred partial pro- and if confirmed, evaluating the additional implementa- tection against experimental ASFV lethal challenge [25, tion of antibody detection methods as performed during 26], therefore demonstrating their protective potential. the eradication campaigns in Spain [42]. China, where the Nonetheless, 25 years later, the exact mechanisms by virus has infected more pigs in only 1 year that in its en- which this partial protection was afforded are not yet fully tire history, might parallel the fastest evolution ever re- understood. While some authors correlated the protection corded for ASFV, therefore increasing the risk for the afforded with the presence of antibody-dependent cell- region to become endemic. What in the short term might mediated cytotoxicity [26], others associated it with the look like a successful exit from the current living drama presence of neutralizing antibodies [27], an issue still con- could bring long-term consequences for global commerce. troversial today [28]. A third complementary mechanism of antibody-mediated protection has been postulated [29] Recombinant LAVs: coming closer to the market based on studies using sera from pigs surviving the infec- Today the possibility of deleting specific genes using re- tion with several hemadsorbing ASFV strains. In these combination technologies [43] or by genome editing using studies, a direct correlation was observed between the the CRISPR-Cas9 system [44], opens the possibility of Bosch-Camós et al. Porcine Health Management (2020) 6:17 Page 4 of 14 obtaining safer LAVs. The main LAVs tested in vivo are The definition of homologous and heterologous for ASF summarized In Table 1, especially remarking those with has not been well defined, observing a lack of cross- potential to protect against the virus currently circulating protection on occasions between viruses isolated in close in Europe and Asia. Genome manipulation has provided endemic regions and in the same period [30]. Although crucial information about essential and non-essential useful to understand ASFV evolution, genotyping [6] does ASFV genes involved in replication, viral morphogenesis not correlate with protection. On the other hand, serotyp- and of course, in virus virulence [4, 65]. ing based on hemadsorbing inhibition assays, seems to As a good example for the purpose of this review, deple- partially explain cross-protection [67], but there is still tion of EP402R gene from the non-pathogenic BA71V iso- much to learn, since protection can be afforded in the ab- late, allowed in vitro identification of CD2v (EP402R sence of CD2v [48, 62, 63]. Furthermore, viruses with product), as the ASFV hemagglutinin [45]. Unfortunately, identical CD2 and lectin behave as heterologous in vivo the non-infectious nature of BA71V made the in vivo [30]. Lack of in vitro correlates for protection leaves ex- characterization of BA71VΔCD2, or any other recombin- perimental in vivo challenging as the only way to demon- ant virus made on this background impossible [66]. The strate the cross-protective nature of a novel vaccine. first deletion mutant tested in vivo was Δ8-DR, a recom- Twenty-five years after its deletion from the non- binant precisely lacking CD2v, made on the virulent pathogenic BA71V [45], the EP402R gene (CD2v) was Malawi Lil-20/1 ASFV strain [46]. Despite its incapability deleted from the parental BA71 virulent virus to obtain to bind red blood cells, Δ8-DR was still lethal in vivo, BA71ΔCD2 for the completion of in vivo studies [48]. showing a delay/reduction in viremia kinetics but causing BA71ΔCD2 induced strong humoral and cellular re- clinical signs and mortality similar to those caused by the sponses and conferred solid protection against the hom- parental virus. From here, few genes have been identified ologous virus (BA71) in a dose-dependent manner. For as implicated in the virulence of different ASFV virulent the first time solid protection was conferred not only strains: DP71L (NL), B119L (9GL), DP96R (UK), DP148R against homologous virus but also against heterologous and multigene families 360 and 505 (MGF360 & viruses such as E75 (Genotype I) or the phylogenetically MGF505) [49–53, 55–57]. Some of these genes have been more distant Georgia2007/1 strain (Genotype II) cur- also eliminated from Georgia2010, the Genotype II virus rently circulating in Continental Europe and Asia. Fi- currently circulating in Europe and Asia; deletions that nally yet importantly, BA71ΔCD2 presents the have caused attenuation and different degrees of homolo- additional advantage of being capable of stably growing gous protection in vivo [54, 58]. Interestingly, the con- in the commercial Cos-1 cell line without needing previ- comitant elimination of the 9GL and UK from the ous adaptation. Despite the impressive protective cap- Georgia2010 virus resulted in more effective vaccine pro- acity achieved with this experimental vaccine, totypes than those lacking individual ORFs [59]. Similarly, BA71ΔCD2 needs further improvement, mainly from recent results published in Science China Life Science, the biosafety point of view. A small proportion of vacci- demonstrate the safety and the efficacy of a new recom- nated pigs show short periods of low, albeit detectable binant LAV obtained from deleting the already described BA71ΔCD2 in both sera and nasal swabs coinciding with MGF360/530 and CD2v virulence factors [64]. It is of cru- mild fever spikes, but no other ASF-compatible clinical cial relevance for beginners in the field to be aware that signs. All animals end up eliminating the virus in the concomitant elimination of ASFV virulence factors does first 4 weeks after vaccination. not always result in better attenuation, on occasions yield- This chapter deserves a closing with unexpected good ing very weak viruses incapable to grow in vivo, or at least news. The specific deletion of the previously uncharac- incapable of inducing protective responses. This is the terized I177L gene provokes the dramatic attenuation of case for example of a Georgia2010 triple mutant lacking the virus and most importantly, confers sterilizing pro- 9GL, UK and NL [61] or a Georgia2010 double mutant tection against the virus currently threating our pig in- lacking 9GL and MGF360/MGF505 [60]. Likewise, deple- dustry [68]. This result clearly demonstrates that the tion of specific virulence factors from natural LAVs, have systematic deletion of each one of the ASFV genes, inde- decreased their ability to protect against challenge with pendent of in silico predictions could bring satisfactory the parental virulent virus [62, 63]. And finally, depletion surprises. This is not a question of luck but conversely, of a single gene can result in different phenotypes depend- rewards one of the few groups consistently working on ing on the virus strain used, as has been demonstrated for ASFV vaccine discovery, a field with plenty of frustra- NL [51], and more recently for CD2v [47, 48]. These ob- tions that also bring some satisfactions. servations are of extreme relevance when rationally de- Despite their imperfections, LAVs available today con- signing LAVs against ASFV, since on most occasions they fer a level of protection against experimental ASF infec- limit their protection against the parental virulent strain tion far better than any other vaccine strategy so far (homologous virus) but not against heterologous strains. tested (see the following chapter). Ideal LAVs should Table 1 Recombinant LAVs tested in vivo, specially indicating those providing protection again the Genotype II virus currently circulating in Asia and Europe (bolded data) Deleted gene(s) Protein(s) Parental ASFV Recombinant LAVs Target cells Reference Strain Virulence Virulence Protection EP402R CD2v BA71V Non-pathogenic Non-pathogenic None Vero [45] Malawi Lil-20/1 Virulent Virulent None Macrophages [46] Georgia2010 Virulent Virulent None Macrophages [47] BA71 Virulent Attenuated BA71, E75, Georgia2007/1 COS-1 & Macrophages [48] Bosch-Camós et al. Porcine Health Management DP71L NL Malawi Lil-20/1 Vrulent Virulent Malawi Macrophages [49, 50] E70 Virulent Attenuated E70 Macrophages [50, 51] B119L 9GL Pretoriuskop/96/4 Virulent Attenuated Pretoriuskop/96/4 Macrophages [52] Malawi Lil-20/1 Virulent Attenuated Malawi Macrophages [53] (2020) 6:17 Georgia2010 Virulent Attenuated Georgia2010 Macrophages [54] DP96R UK E70 Virulent Attenuated E70 Macrophages [55] DP148R Benin Virulent Attenuated Benin Macrophages [56] MGF360 & MGF505 Benin Virulent Attenuated Benin Macrophages [57] Georgia2010 Virulent Attenuated Georgia2010 Macrophages [58] B119L & DP96R 9GL & UK Georgia2010 Virulent Attenuated Georgia2010 Macrophages [59] MGF360 & MGF505 & B119L Georgia2010 Virulent Highly attenuated None Macrophages [60] B119L & DP96R & DP71L 9GL & UK & NL Georgia2010 Virulent Highly attenuated None Macrophages [61] DP71L & DP96R NL & UK OUR T88/3 Attenuated Highly attenuated None Macrophages [62] A238L NFAT regulator NH/P68 Attenuated Highly attenuated L60 COS-7 & Macrophages [63] A224L Apoptosis inhibitor EP153R MHC-I antigen presenting modulator MGF360 & MGF505 & EP402R MGF505-1R, MGF505-2R, China HLJ/18 Virulent Attenuated China HLJ/18 Macrophages [64] MGF505-3R, MGF360-12 L, MGF360-13 L, MGF360-14 L & CD2v Page 5 of 14 Bosch-Camós et al. Porcine Health Management (2020) 6:17 Page 6 of 14 however accomplish the following requisites before be- The Yin and the Yang of ASFV-specific antibodies coming a reality in the field: The ASFV serological determinants determined so far are listed in Table 2, specifically indicating those capable 1) Safety. Experimental LAVs should demonstrate in of inducing antibody responses after immunization the field not only to be efficacious, but also safe for in vivo. As described above, the presence of neutralizing the vaccinated pigs, for the in-contact animals and antibodies and their potential role in protection is still for the environment. today polemic [28]. On the one hand, neutralizing 2) DIVA. Vaccinated pigs should ideally induce an monoclonal antibodies recognizing the major capsid immune response distinguishable from naturally protein p72 were described decades ago [76], but no fur- infected pigs. ther development has been recorded. Similarly, specific 3) Biofactory. Vaccines should be produced under polyclonal antibodies against p54 (encoded by the E183L high quality standards and following the policy of ORF) and p72 (B646L) were capable of blocking the the corresponding regulatory agencies, thus ASFV-attachment to susceptible cells, while antibodies requiring stable cell lines for producing the LAV at against p32 (CP204L) inhibited virus internalization, a large scale. both mechanisms essential to protect pigs against E75 4) Cross-protection. Cross-protective vaccines could lethal challenge [27]. Unfortunately, the protection be useful not only to protect a specific region against afforded by p72, p54 and p32 was not reproducible using a single virus, but against many different viruses from other infectious model systems [77], opening new con- the same genotype or from different genotypes cerns about their relevance in protection and contribut- (ideally all), thus covering endemic regions of Africa ing to feed the controversy still in force, about the where up to 24 genotypes have been described. protective role of antibody-mediated neutralization. Due 5) Wildlife: LAVs formulations compatible with wild- to the fact that p72, p32 and p54 are among the most life immunization campaigns will also be demanded immunogenic structural proteins, they have been in- if willing to control and eradicate ASF. Bait-vaccine cluded in diverse experimental vaccine formulations formulations should also demonstrate their safety aiming to confer protection against several virulent and efficacy in field studies. ASFV isolates, with little if any significant effect [37, 38, 72, 73, 77]. Vaccines are one of the best human inventions, but Parallel studies performed aiming to identify ASFV this truth circumscribes exclusively the “good ones”: proteins responsible of inducing hemagglutination in- those proven efficient, safe and correctly administered in hibitory antibodies [30], provided the identification of the field. Balancing the risks and the benefits of imple- CD2v as the ASFV hemagglutinin. Thus, immunization menting each available vaccine, applying scientific cri- of pigs with Sf9 insect cells infected with a recombinant teria and avoiding pre-established interests and baculovirus encoding the ASFV protein CD2v in the prejudices, should be mandatory in the future. presence of Freund’s adjuvant, conferred protection against E75 ASFV challenge. The protection afforded Antigen discovery and development of subunit vaccines was dose-dependent and correlated with the presence of In order to avoid, or at least diminish, the unwanted side antibodies capable of inhibiting hemagglutination and effects of traditional inactivated and attenuated vaccines, the homologous ASFV infection in vitro [29]. This work subunit vaccines arise as an ideal option for the future has been more recently confirmed by identifying CD2v development of ASF vaccines [15]. As mentioned above, and the ASFV lectin as key protective antigens [83], little is known about the mechanisms involved in protec- which could be useful to serotype ASFV [67]. As de- tion and even less about the antigens responsible of such scribed for p32, p54 and p72, some experimental vaccine protection. To fill these gaps, research from both public formulations containing CD2v have failed at inducing administrations and the private sector should be encour- solid protection, perhaps reflecting antigenic and patho- aged: from antigen discovery to adjuvant and expression genic differences between the ASFV strains used or in vector, more research investment is needed to design the vaccine formulations per se [37, 38, 78, 81, 84]. the ideal vaccines of the future. Screening of potential Together with p54, p32 and p72, many other antigens antigenic determinants within the ASFV is a challenging have been identified by sera from recovered pigs as immu- task due to the complex nature of this virus, encoding nodominant antigens, including the structural polypro- more than 150 ORFs and from which many of them tein pp62 [80]. Sera from convalescent pigs also recognized there is neither expression nor functional data available. some other structural proteins (A104R, C-type lectin In this section, we collect relevant studies regarding pEP153R, chaperone pB602L and K78R), non-structural ASFV antigen discovery as well as significant advances proteins (RNA reductase, DNA ligase and thymidine kin- in the development of subunit vaccines against ASF. ase) as well as proteins of unknown function and location Bosch-Camós et al. Porcine Health Management (2020) 6:17 Page 7 of 14 Table 2 ASFV serological immunodeterminants, indicating those described to be recognized by sera from pigs recovered from ASFV infection (antigenic) or to induce immune responses after in vivo immunization (immunogenic) Gene name Protein Antigenic Reference Immunogenic Reference Structural A104R Histone-like DNA-binding protein Yes [69, 70] Yes [71] A137R P11.5 – – Yes [72] B438L p49 – – Yes/Low [73, 74] B602L p72 chaperone Yes [69, 70] Yes [74, 75] B646L p72, major capsid protein Yes [27, 69, 76] Yes [71, 72, 77, 78] CP204L p30 Yes [27, 69] Yes [38, 71–73, 77–79] CP2475L pp220 – – Yes [72] CP2475L/partial p37 – – Yes [71] CP530R pp62 Yes [80] Yes [38, 81] CP530R/partial p35 – – Yes [37, 78] CP530R/partial p15 – – Yes [37, 78] D117L Major transmembrane – – Yes [73] protein p17 E183L p54 Yes [27, 69, 70] Yes/Low [37, 71, 73, 77–79] EP153R C-type lectin – – Yes [73] EP402R HA/CD2v Yes [82] Yes [71, 73, 78] K78R p10, DNA-binding Yes [69] – – KP177R p22 – – Yes/Low [73, 77] E120R p14.5 – – Low [73] H108R Inner envelope – – Low [73] O61R p12 – – Yes [73] Non-structural B119L 9GL, virus assembly – – Yes [74] F334L RNA reductase Yes [69] – – I215L Ubiquitin conjugating enzyme – – Yes [38] K196R Thymidine kinase Yes [69] – – L10L KP177R-related – – Yes [73] NP419L DNA ligase Yes [69] – – Uncharacterized A151R – – – Yes [74] C129R – – – Yes [38] C44L – Yes [69] – – CP312R – Yes [69] Yes [78] E184L – Yes [69] – – K145R – Yes [69] – – K205R – Yes [69, 70, 75] Yes [74] M448R – – – Yes [38] Multigene families MGF110-4 L XP124L – – Yes [38] MGF110-5 L V82L – – Yes [38] Bosch-Camós et al. Porcine Health Management (2020) 6:17 Page 8 of 14 (C44L, CP312R, E184L, K145R and K205R) [69, 70, 75, 85]. that cellular responses play in protection. This reality Unfortunately, the protective efficacy of most of these anti- has dramatically changed in recent years, and future vac- gens has not been confirmed. cinologists would have to focus their attention also on More recently, immunization of pigs with complex this crucial arm of the immune response. However, few ASFV-antigen formulations induced specific antibody re- in vitro studies have centered their attention on identifi- sponses that allowed the identification of additional anti- cation of ASFV proteins inducing CTLs. The antigens so genic determinants. Thus, Babraham syngeneic pigs far recognized by ASFV-specific T-cells, as well as those immunized with a cocktail of 44 antigens in a DNA-prime described as immunogenic in vivo (capable to induce recombinant vaccinia-boosting regime, recognized D117L specific T-cell responses upon immunization), are listed as a novel ASFV immunogenic protein [73]. After chal- in Table 3. lenge with Georgia2007/1, all pigs developed acute ASF, The first ASFV CTL target identified in vitro was pro- albeit viral genome levels were significantly reduced in im- tein p32 [87], followed by the major capsid protein p72 munized pigs in blood and lymphoid tissues. More re- and G1340L [86, 92]. Aiming to maximize the screening cently, adenovirus-priming and vaccinia Ankara (MVA)- coverage of ASFV antigens, a random plasmid library of boosting regimes using diverse antigen combinations, ASFV genomic DNA was generated and expressed in allowed the definition of five additional ASFV antigens as pig-derived fibroblasts. Sequencing of the clones that good antibody inducers: MGF110-4 L, MGF110-5 L, stimulated CD8+ T-cell proliferation revealed that one of M448R, C129R, I215L [38]. As before, immunization did the clones encoded a segment of I329L, a putative ASFV not protect pigs from acute ASF (OUR T88/1 challenge) membrane protein. Notably, this technique allowed the but reduced the viremia in a proportion of outbred pigs. identification of sequences that were in different reading Immunization with different cocktails of recombinant frames than any of the known ASFV ORFs [90], indicat- adenoviruses allowed the confirmation of the immuno- ing that the proteome of ASFV could be more complex genicity of B602L, K205R-A104R, CP530R, CP2475L and than previously believed. This hypothesis has been re- the extracellular domain of EP402R, identifying for the cently supported by the identification of novel ASFV first time A151R and B119L as good antibody inducers, ORFs by RNA sequencing methods [93]. albeit none of the cocktails induced significant protec- Despite the unarguable reality of CD8+ T-cells playing tion against Georgia2007/1 [71, 74, 81]. Immunization a role in protection, few ASFV antigens have demon- with recombinant adenoviruses encoding: p32, p54, strated their protective potential (Table 4), still far from pp62, p72, and p37 and p150 (mature proteins processed that afforded by LAVs. Today we know that CD2v can from the pp220 polyprotein) seemed to afford some pro- also exhort its protective effects by stimulating the in- tection depending on the adjuvant used, while other vac- duction of specific CD8+ T-cells [88, 89]. Thus, DNA cine formulations exacerbated the disease [81]. immunization with a plasmid encoding p32, p54 and the Despite the efforts made, none of these antigen-specific extracellular domain of CD2v, fused to ubiquitin aiming antibodies have been confirmed to be protective in an un- to enhance the CTL responses induced, conferred partial arguable manner and we are still far from identifying protection against lethal challenge with E75. The protec- ASFV antigens capable to confer solid protection against tion afforded correlated with the induction of specific Georgia2007/1 challenge and even further from the opti- CD8+ T-cells recognizing two 9-mer peptides from the mal formulations. In fact, antigenicity and protection do CD2v and in the absence of antibodies [89]. Work per- not concur and, on occasions, and an inverse correlation formed in our laboratory using DNA immunization has has even been demonstrated. This effect has been de- allowed not only the identification of new protective anti- scribed upon natural and experimental ASFV infection gens but has also contributed to dissect the mechanisms with LAVs and also after immunization with inactivated involved in protection. Thus, immunization with a ran- and subunit vaccine formulations [17, 36–38, 81]. dom library containing more than 4000 clones encoding The fine balance existing between the protective and random ASFV genome fragments (excluding the se- detrimental role of ASFV-specific antibody response quences encoding p32, p54 and CD2v to avoid competi- needs further investigation, both from the basic and ap- tion) fused to ubiquitin protected 60% of the animals plied point of view. Antibodies play a role in protective against E75 lethal challenge. The protection was again immunity against ASFV, although the specific antigens afforded in the absence of detectable specific antibodies and/or the way to be targeted are still unknown. after immunization and correlating with the induction of ASFV-specific T-cell responses [91] against several anti- The unknown: ASFV antigens inducing T-cell responses gens, including CP312R [94]. These results clearly demon- ASFV vaccinology, as well as traditional veterinary vacci- strated the presence of additional protective CD8+ T-cell nology in general, has focused on triggering antibody re- determinants within the ASFV genome. These results sponses, while little attention has been paid to the role highlighted the convenience of formulating vaccines a la Bosch-Camós et al. Porcine Health Management (2020) 6:17 Page 9 of 14 Table 3 ASFV-specific T-cell determinants, indicating those described to be recognized by PBMCs from pigs recovered from ASFV infection (antigenic) and those inducing T-cell responses after in vivo immunization (immunogenic) Gene name Protein Antigenic Reference Immunogenic Reference Structural B602L p72 chaperone – – Yes [73] B646L p72 Yes [38, 86] Yes [38, 71, 73] CP204L p30 Yes [38, 87] Yes [38, 71, 73] CP2475L pp220 – – Yes [73] CP530R pp62 Yes [38] Yes [38, 71, 73] CP530R/partial p15 – – Yes [37] D117L – – – Low [73] E183L p54 – – Yes [71, 73] E248R Transmembrane myristoylated protein Yes [38] – – EP153R C-type lectin Yes [88] Yes [73] EP402R HA/CD2v Yes [88] Yes [73, 89] H108R Inner envelope – – Low [73] I239L – Yes [90] – – K78R p10 Yes [38] No [38] KP177R p22 Yes [38] Yes [73] B438L p49 – – Yes [73, 74] E199L j18L – – Yes [73] O61R p12 – – Yes [73] Non-structural A179L – – – Low [73] B119L 9GL, virus assembly – – Yes [74] C475L Poly(A) polymerase Yes [38] – – C962R DNA primase Yes [38] – – DP71L Protein phosphatase 1 regulator – – Low [73] E165R dUTPase Yes [38] Yes [38] E296R AP endonuclease Yes [38] – – F1055L Helicase – – Yes [73] F334L RNA reductase Yes [38] – – G1211R DNA polymerase – – Yes [73] G1340L VACV A7 early transcription factor large subunit-like Yes [86] Low [73] H339R Alpha-NAC binding protein Yes [38] – – H359L RNA polymerase subunit 3–11 Yes [38] – – I215L Ubiquitin conjugating enzyme Yes [38] Yes [38] I329L TLR inhibitor – – Low [73] NP1450L RNA polymerase subunit 1 – – Yes [73] NP419L DNA ligase Yes [38] Yes [73] NP868R mRNA-capping enzyme Yes [38] – – O174L DNA polymerase X Yes [38] – – Multigene families MGF110-1 L – Yes [38] Yes [38] MGF110-2 L – – – Low [73] MGF110-4 L XP124L Yes [38] Yes [38] Bosch-Camós et al. Porcine Health Management (2020) 6:17 Page 10 of 14 Table 3 ASFV-specific T-cell determinants, indicating those described to be recognized by PBMCs from pigs recovered from ASFV infection (antigenic) and those inducing T-cell responses after in vivo immunization (immunogenic) (Continued) Gene name Protein Antigenic Reference Immunogenic Reference MGF110-5 L V82L Yes [38] Yes [38] MGF300-1 L J268L Yes [38] Low [73] MGF300-2R – – – Low [73] MGF360-11 L KP362L – – Yes [73] MGF360-15R – – – Low [73] MGF360-16R – Yes [38] – – MGF360-18R – – – Low [73] MGF360-1 L KP360L – – Yes [73] MGF505-11 L – Yes [38] – – MGF505-4R – – – Yes [73] MGF505-5R A498R Yes [73] Uncharacterized 285 L – Yes [38] – – A151R – Yes [38] Yes [38] B407L – – – Low [73] C129R – Yes [38] Yes [38] C257L – Yes [38] CP312R – Yes [38] Yes [38] CP312R – – – Yes [31, 91] D339L – – – Low [73] DP238L – Yes [38] – – E120R – – . Low [73] E146L – Yes [38] Yes [38] E184L – Yes [38] Yes [38] EP364R – – – Yes [73] F317L – – – Yes [73] I243L – Yes [38] – – I73R – Yes [38] No [38] K205R – Yes [38] Yes [73, 74] L8L – Yes [38] – – L10L – Yes [38] Low [38, 73] M1249L – Yes [38] Low [73] M448R – Yes [38] Yes [38] Complement (183875–184,183) – – – Low [73] carte, encoding not only the appropriate ASFV antigens [38]. The specific responses induced were measured in an but also targeting them to specific antigen presentation IFNγ ELISpot assay, identifying peptides from 38 ASFV pathways aiming to enhance the induction of protective proteins in at least one of the tested pigs, including p72 responses and avoiding undesired adverse immune re- and p32, but also many other uncharacterized proteins sponses after vaccination [36]. (Table 3). More recently, an overlapping peptide library comprising Interestingly, immunization with adenoviruses encod- almost 4000 20-mers peptides overlapping by 10 amino ing the structural proteins: p32, p54, pp62, p72 and acids from 133 different ASFV ORFs, was used in in vitro pp220 in the presence of the ZTS-01 adjuvant induced stimulation protocols using PBMCs from ASFV- lower antibody responses and slightly better protection convalescent pigs to identify potential CD8+ T-cell targets (non-statistically significant), than with the BioMize Bosch-Camós et al. Porcine Health Management (2020) 6:17 Page 11 of 14 Table 4 ASFV subunit vaccines tested in vivo that induced some level of protection Gene Name Immunization Challenge Outcome Haplotype Reference strain A179L, B407L, B438L, B602L, B646L, Complement (183875– DNA prime + Georgia No surviving pigs Babraham [73] 184,183), CP204L, CP2475L, CP530R, D117L, D339L, DP71L, rVACV boost 2007/1 Reduced viremia and viral loads in E120R, E183L, E199L, EP153R, EP364R, EP402R, F1055L, some tissues F317L, G1211R, G1340L, H108R, I329L, K205R, KP177R, L10L, M1249L, MGF110-2 L, MGF110-4 L, MGF300-2R, MGF360-11 L, MGF360-15R, MGF360-18R, MGF360-1 L, MGF505-4R, MGF505-5R, NP1450L, NP419L & O61R B646L, CP204L, CP2475L (p37, p150), CP530R & E183L rAd Georgia Partial protection: 5/9 surviving pigs Outbred [71, 81] 2007/1 versus 1/5 control pigs Reduced viremia depending on the adjuvant used Pigs recover from infection Authors claim that none of the immunogens conferred statistically significant protection A151R, B646L, C129R, CP204L, CP530R, E146L, I215L, I73R, rAd prime + OUR T88/ No surviving pigs Outbred [38] L8L, M448R, MGF110-4 L & MGF110-5 L MVA boost 1 3/6 pigs with reduced and delayed clinical signs, 5/6 pigs with reduced viremia Reduced viral load in some tissues CP2475L & A137R Synthetic E70 No surviving pigs Outbred [72] peptides Increased average survival Reduced mean viral titers CP204L & B646L Synthetic E70 No surviving pigs Outbred [72] peptides Increased average survival Reduced mean viral titers CP204L, E183L & EP402 DNA (as a E75 Partial protection: 2/6 surviving pis Outbred [89] fusion with Delayed clinical signs and viremia Ub) Pigs recover from the infection CP312R DNA E75 Partial protection: 6/10 surviving Outbred [91, 94] expression pigs library Reduced virus titers in blood, shedding and lesions Pigs recover from the infection 0226 adjuvant, reflecting most probably the differential in- As a practical observation, we confirm ASFV in vivo nate immune responses induced by each adjuvant [81]. challenge as the only valid method for evaluating the On the other hand, immunization with cocktails up to 47 protective potential of an experimental vaccine. Lack of antigens in a DNA priming and vaccinia virus-boosting re- correlates of ASFV protection and protective targets, gime, allowed the identification of novel T-cell antigens. precludes assuming the potency of a given vaccine for- Unfortunately, none of the pigs survived the lethal chal- mulation based on the immune response induced. lenge (Table 4) albeit some pigs showed a significant re- Although the use of subunit vaccines based in ASFV duction of virus titers in blood and lymph nodes [73]. fragments overcome most of the risks inherent to LAVs, Finally, priming pigs with a cocktail of replication defi- their use in the field is far from becoming a reality due to cient adenoviruses encoding 12 ASFV ORFs and boost- the low efficacy demonstrated so far. As mentioned above, ing with same antigens encoded in MVA, allowed the lack of profound knowledge of the protection mechanisms confirmation of a collection of antigens as capable of in- together with the absence of known protective antigens ducing specific T-cell responses and to partially protect have complicated the design of efficient subunit vaccines pigs from challenge with OURT88/1 (Table 4). Although against ASFV. These are in our understanding the future all the animals had to be sacrificed before day 8 after directions in research and development: challenge, three out of six pigs showed delayed onset of clinical signs as well as reduced viremia. Interestingly, 1- Correlates of protection. Easy vaccines have immunization with a second collection of immuno- already been designed. ASFV is a complex virus gens provoked the exacerbation of the disease, coin- capable of inhibiting the pig immune system by ciding with the induction of weaker CD8+ T-cell interfering with multiple pathways. Understanding responses [38]. ASFV strengths will teach us about its weakness. Bosch-Camós et al. Porcine Health Management (2020) 6:17 Page 12 of 14 Optimal vaccination should not only enhance be envisioned as the future choice, based on knowledge protective responses but also avoid prejudicial and scientific evidence. immune responses. ASF is a global problem that we were incapable of 2- Antigen discovery. Both humoral and cellular stopping. We should learn from our mistakes and find responses can be protective. Identifying as many global solutions. antigens and/or epitopes from the whole proteome Acknowledgments is absolutely needed. This has to be performed in We thank Kevin Dalton for final editing of the manuscript. We also thank parallel with understanding the mechanisms Jordi Argilaguet, Francesc Accensi, Sonia Pina, Jinya Zhang and Uxía Alonso involved in protection. for their contribution to this manuscript, María Jesús Navas for her technical support and personnel at CReSA-BSL3 animal facilities for their continuous 3- Antigen delivery. We need to know not only what help. Finally, our warmest recognition to Francisco Ruiz-Gonzalvo, our to induce and with which antigen, but also how to mentor. deliver it in an efficient manner and at an affordable price. Vaccinology will dramatically change in the Authors’ contributions EL and LBC performed most of the bibliographic search on LAVs and subunit future with the invention of new expression vectors vaccines respectively. LBC prepared the Tables 2, 3 and 4 here presented. FR that should be approved by the safety agencies. prepared Table 1, coordinated and edited the text writing. The three authors 4- Adjuvants. Little attention is given to the need of contributed to the writing and revised the submitted version of the manuscript. EL and LBC finished their thesis in the lab last year working in novel and efficient adjuvants to formulate a la carte LAVs and subunit ASF vaccine development, respectively under the vaccines, capable of inducing exclusively what is coordination of FR (PI of the ASF research line at IRTA-CReSA, Barcelona). The desired. Adjuvants could be an essential tool to authors read and approved the final manuscript. achieve this goal. Funding 5- Others. DIVA concept, bait-vaccines and cross- This study was supported by the Ministry of Science, Innovation and protection. Subunit vaccines against ASFV will be Universities from the Spanish Government (grant number AGL2016–78160- C2–1-R). The authors are also grateful to the Centres de Recerca de DIVA by themselves with many immunodominant Catalunya (CERCA) Programme and Global Alliance for Research on African antigens available for differential diagnostics. Cross- swine fever (GARA). protection however is an issue difficult to tackle today since we need to identify first the mecha- Availability of data and materials Not applicable. nisms and the antigens involved. Similarly, bait- vaccines for oral immunization of wild pigs is an Ethics approval and consent to participate utopia today, at least for ASF-subunit vaccines, al- References cited justified the corresponding ethic statement at the time of publishing. beit it has been recently proposed to experimentally In vivo experiments performed in the biosafety level 3 plus (BSL3+) facilities immunize wild boars with a naturally attenuated at the Centre de Recerca en Sanitat Animal (IRTA-CReSA; Barcelona, Spain). ASFV strain [41]. We should first identify the anti- Animal care and procedures were carried out in accordance with good experimental practices and under the supervision of the Ethical and Animal gens to incorporate, and then know how to formu- Welfare Committee of the Institut de Recerca en Sanitat Animal (IRTA). late them in order to afford significant protection According to European (Directive 2010/63/EU of the European Parliament against ASF. We are not close of having a subunit and of the Council of 22 September 2010 on the protection of animals used for scientific purposes) and Spanish (Real Decreto 53/2013) normative. vaccine, but if we do not invest in research today, Not applicable. they will never become a reality. Consent for publication Conclusions Not applicable. ASFV vaccines are closer than they appear. On one hand, Competing interests several experimental prototypes based on LAVs are ma- The authors declare that the research was conducted in the absence of any ture enough to be transferred to the private sector for commercial or financial relationships that could be construed as a potential conflict of interest. registration and field trials. International agencies, govern- ments, industry and academy should join together to dis- Received: 10 March 2020 Accepted: 6 May 2020 cuss the opportunity of their implementation in very specific scenarios, taking into account the benefits versus References the prejudices that the new LAVs can provide and the dif- 1. Alejo A, Matamoros T, Guerra M, Andrés G. A proteomic atlas of the African ferent sensitivity for the ASF problem around the globe. swine fever virus particle. J Virol. 2018;92(23):e01293–18. Parallel efforts should be made in research and devel- 2. Liu S, Luo Y, Wang Y, Li S, Zhao Z, Bi Y, et al. 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Vaccination with viral protein-mimicking peptides postpones mortality Publisher’s Note in domestic pigs infected by African swine fever virus. Mol Med Rep. 2011; Springer Nature remains neutral with regard to jurisdictional claims in 4(3):395–401. published maps and institutional affiliations.