Adjuvant-induced macrophage activation compromises BA71ΔCD2-mediated protection against African swine fever virus

Adjuvant-induced macrophage activation compromises BA71ΔCD2-mediated protection against African swine fever virus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Adjuvant-induced macrophage activation compromises BA71ΔCD2-mediated protection against African swine fever virus Aida Tort-Miró, Sergio Montaner-Tarbes, David Marín-Moraleda, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7815447/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Apr, 2026 Read the published version in npj Vaccines → Version 1 posted 11 You are reading this latest preprint version Abstract While the development of effective subunit vaccines against African swine fever (ASF) is ongoing, live attenuated vaccines (LAVs) remain the only current strategy capable of inducing robust protective immunity. However, potential biosafety concerns limit their implementation in the field. Thus, further research is required to develop optimized LAVs with better biosafety profiles. Both the ASF virus (ASFV) and derived LAVs suppress innate immune responses of macrophages, thereby limiting their contribution to the induction of protective immune responses. We hypothesized that adjuvants could restore the functionality of LAV-infected macrophages, allowing for a reduction in the vaccine’s effective dose and consequently minimizing the risk of adverse events. To test this hypothesis, we intranasally vaccinated pigs with a suboptimal dose of the LAV BA71ΔCD2, either alone or in combination with two adjuvants derived from the immunostimulatory bacterium Rothia nasimurium . The two immunostimulants enhanced the responsiveness of BA71ΔCD2-infected macrophages, which acquired features of antigen presenting cells. However, both adjuvants reduced the levels of ASFV-specific humoral and cellular responses induced by BA71ΔCD2, consequently decreasing the level of protection against a lethal challenge. Further in vitro analyses demonstrated that adjuvant-activated macrophages acquired an antiviral state, thereby reducing the replication capability of the LAV. Thus, the adjuvant-mediated decline in vaccine efficacy might result from a lower antigen production by infected cells. These results demonstrate that the use of adjuvants combined with ASFV-based LAVs will require a fine-tune manipulation of macrophages, enhancing their functionality while avoiding a significant inhibition of virus replication, reaching the required balance between the levels of viral antigens and innate immune responses to trigger a protective adaptive immunity. Biological sciences/Immunology Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The development of a safe and effective African swine fever (ASF) vaccine remains a major global challenge. The disease is one of the biggest threats affecting the pig industry worldwide, with a complex epidemiological pattern in Africa, and currently spreading in Asia and Europe through the infection of domestic pigs and wild boars ( https://efsa.onlinelibrary.wiley.com/doi/ 10.2903/j.efsa.2023.8016 ). While the development of subunit vaccines is progressing 1 , 2 , it continues facing important limitations due to the lack of knowledge on protective antigens and immune responses 3 , 4 . In contrast, several experimental live attenuated vaccines (LAVs) confer robust immunity and protection against lethal challenge 5 . However, biosafety concerns hold back their implementation in the field 6 . Indeed, two LAVs were approved in Vietnam in 2022 7 , but their widespread usage is not yet a reality due to the potential of reversion to virulence and the emergence of new recombinant strains 8 – 11 . Thus, while waiting for the generation of safe and effective subunit vaccines, the development of novel LAV-based strategies with enhanced biosafety profiles might boost their application in ASF endemic areas. The main target of ASF virus (ASFV) are monocytes/macrophages. With more than 150–200 encoded proteins 12 , the virus has a high capability to hijack the infected cells, modulating their functional responses and eventually causing fatal cytopathic effects 13 . Indeed, throughout its replication cycle, ASFV modulates cytokine production, antigen presentation pathways and apoptosis- or pyroptosis-programmed cell death in infected cells 14 , 15 . Consequently, macrophages are disabled to act as antigen presenting cells (APC), not providing to other immune cells the required signals for a proper induction of a protective adaptive immune response. To note, many LAVs or naturally attenuated ASFV strains inducing protective immunity trigger higher innate immune responses in infected macrophages than the parental virulent strains 14 , 16 , 17 . This indicates that protective immunity induced by live ASFV-based immunisation is associated with the preservation of some degree of functionality in infected macrophages. However, the balance between virus attenuation and immunogenicity is subtle, and the generation of LAVs achieving a trade-off between biosafety and efficacy is complex. Notably, attempts to obtain safer LAVs through multiple gene deletion quite often result in reduced immunogenicity and loss of protective immunity 18 – 23 . The use of adjuvants has usually been restricted to subunit or inactivated vaccines, which commonly have a weak intrinsic capability to stimulate immune responses 24 . In contrast, LAVs have an inherent ability to activate the innate immunity upon their recognition by pattern recognition receptors (PRRs), thus promoting the induction of vaccine-specific adaptive immune responses 25 . However, a few experimental studies have evaluated the co-administration of LAVs with adjuvants to test their ability to improve vaccine efficacy. In some cases, the combination of adjuvants with live vaccines has shown positive results, such as reduced vaccine side effects following vaccination with live Leishmania major combined with CpG DNA 26 , 27 , and sterilising immunity against pathogenic simian human immunodeficiency virus (SHIV) in macaques vaccinated with a live attenuated SHIV expressing the adjuvant Ag85B 28 . However, further research is required to evaluate the potential use of adjuvants for LAVs, since the effects will be highly adjuvant- and context-dependent. For instance, while the adjuvant a-C-galactosylceramide improved immune responses and protection in mice vaccinated with live attenuated influenza virus 29 , another study in pigs showed that the closely related adjuvant a-galactosylceramide interfered with heterotypic cross-protective immune responses 30 . Here, we hypothesised that adjuvant-mediated activation of macrophages during ASF LAV immunisation would enhance vaccine efficacy, allowing for the reduction of the vaccine dose required to induce a protective immunity, and consequently improving its biosafety. To test this, pigs were intranasally immunised with a suboptimal dose of the ASF BA71ΔCD2 vaccine prototype, which confers partial protection against a lethal challenge 31 . As adjuvants, we used two different formulations derived from Rothia nasimurium , a bacterium with immunostimulatory properties that robustly activates porcine alveolar macrophages 32 . The results demonstrated that despite the enhanced responsiveness of macrophages induced by these adjuvants, their administration with the BA71ΔCD2 did not improve vaccine immunogenicity and efficacy in vivo . Indeed, the use of the adjuvants impaired vaccine-induced humoral and cellular responses, and the degree of protection against a challenge with a virulent ASFV strain. Importantly, further in vitro analyses showed a lower replication capacity of BA71ΔCD2 in adjuvant-activated macrophages, indicating that the decreased vaccine effectiveness might result from a reduction in the antigen levels available to induce a protective immunity. These results represent a step forward in the understanding of early processes underlying LAV-induced immune protection, which might eventually help to rationally improve their biosafety profile. Results 1. Rothia nasimurium -derived immunostimulants overcome the lack of responsiveness of BA71ΔCD2-infected macrophages . We have recently characterized the immunostimulatory properties of heat-inactivated Rothia nasimurium (HI-Ro), demonstrating its capability to activate antigen presenting cells, thus showing its potential as a vaccine adjuvant 32 . Here, aiming to obtain a more purified product, we investigated whether the immunostimulatory components were present in cell-free supernatants from R. nasimurium culture. Indeed, stimulation of porcine alveolar macrophages (PAMs) with supernatants induced IFN-γ, TNF (Fig. 1 a-c), IL-12, and IL-1β ( Supplementary Fig. 1 ) secretion. Notably, the stimulatory capacity was only observed in a fraction of the supernatant containing components with a molecular weight > 100kDa (Frac-Ro), and not in fractions containing smaller components (Fig. 1 a-c and Supplementary Fig. 1 ). Interestingly, in contrast to Frac-Ro, HI-Ro did not stimulate IFN-γ production in macrophages (Fig. 1 a), while both products induced robust TNF production (Fig. 1 b). The stimulatory capacity of Frac-Ro was further demonstrated by analysing the transcriptome of treated macrophages by RNA-sequencing. Gene Ontology (GO) analysis of differentially expressed genes showed an enrichment in terms related to innate immunity, such as NF-κβ, MAPK, TLR and C-type lectin signalling pathways (Fig. 1 c). Despite the differences mentioned above regarding IFN-γ production, this transcriptomic signature was very similar to HI-Ro- and LPS-treated macrophages 32 , upregulating genes encoding for cytokines and chemokines, factors involved in inflammatory pathways, activation markers and receptors, and interferon stimulated genes (Fig. 1 d). Next, we evaluated whether HI-Ro or Frac-Ro treatment of macrophages infected with the ASFV vaccine prototype BA71ΔCD2 can overcome the viral induced immunosuppressive status, thus enhancing their functionality as antigen presenting cells. BA71ΔCD2-infected PAMs were stimulated with HI-Ro or Frac-Ro, and compared to IFN-γ or LPS, and their responsiveness was analysed by cytokine production and transcriptomic analysis. As previously demonstrated in macrophages infected with the highly virulent ASFV strain Georgia2007/1 32 , BA71ΔCD2 infection likewise failed to induce cytokine production in PAMs. However, this lack of responsiveness was overcome in HI-Ro and LPS-treated cells, which robustly secreted TNF. Stimulation with Frac-Ro and IFN-γ showed a similar but not statistically significant tendency (Fig. 2 a). In contrast, IFN-γ production was only induced in BA71ΔCD2-infected cells following Frac-Ro stimulation, showing similar results with IFN-γ, but not statistically significant (Fig. 2 b). To note, IL-1β was secreted only in HI-Ro- and LPS-treated macrophages although this effect did not reach statistical significance (Fig. 2 c). Finally, the expression levels of 24 genes related to innate immunity were upregulated upon HI-Ro-stimulation of BA71ΔCD2-infected PAMs compared with non-treated and infected or non-infected cells (Fig. 2 D; Supplementary Data 1 ). In contrast, Frac-Ro only significantly increased the expression of a few genes such as CCL3 , CCL4 and TNF . Taken together, these results indicate that the immunosuppressive state induced in ASFV-infected macrophages can be at least partially rescued by macrophage-activating components from R. nasimurium , suggesting their potential as adjuvants for ASFV-based LAVs. 2. The use of HI-Ro or Frac-Ro as adjuvants impairs BA71ΔCD2-induced immunogenicity in pigs. Intranasal vaccination with BA71ΔCD2 confers total or partial protection against a lethal challenge depending on the vaccine dose ( https://pubmed.ncbi.nlm.nih.gov/36350837/ ). Thus, to evaluate the adjuvant potential of HI-Ro or Frac-Ro, we combined each of them with a suboptimal single dose of BA71ΔCD2 (10 4 pfu) (Fig. 3 a). The use of adjuvants did not modify the safety profile of the BA71ΔCD2 vaccine prototype. Indeed, all vaccinated pigs showed lack of clinical signs, regardless of the use of adjuvants, with rectal temperatures below 41ºC and low clinical scores as in unvaccinated pigs ( Supplementary Fig. 2a-b ). Consistently, virus loads in serum and nasal swabs were negative except for one animal vaccinated without adjuvant showing low levels of virus ( Supplementary Fig. 2c-d ). To evaluate the effect of using HI-Ro or Frac-Ro as adjuvants, we next analysed cellular and humoral ASFV-specific immune responses induced post-vaccination (p.v.) (Fig. 3 a). Importantly, although between-group differences did not reach statistical significance, point estimates for IFN-γ-producing cells and IgG titres at day 22 p.v. were lower with HI-Ro or Frac-Ro (Fig. 3 b-c; Supplementary Fig. 3a ). Indeed, both ASFV-specific IgG1 and IgG2 antibody titres (Fig. 3 d; Supplementary Fig. 3b-c ), as well as ASFV-specific IgM titres at 14 days p.v. (Fig. 3 e; Supplementary Fig. 3d ), were lower in the two groups vaccinated with adjuvants. Finally, a correlation analysis between ASFV-specific IFN-γ-producing cells and IgG titres at day 22 p.v. demonstrated that pigs responding to vaccination concomitantly triggered both cellular and humoral responses, regardless of the vaccination group (Fig. 3 f). Overall, these results indicate that the use of HI-Ro or Frac-Ro as BA71ΔCD2 adjuvants dampens vaccine-induced immunity, equally affecting both systemic cellular and humoral responses. 3. Combination of BA71ΔCD2 with HI-Ro or Frac-Ro reduces vaccine efficacy against a lethal ASFV infection. To evaluate the protection induced by BA71ΔCD2 combined with HI-Ro or Frac-Ro, we next challenged the pigs at 22 days p.v. with a lethal intranasal inoculation of the virulent Georgia2007/1 strain (Fig. 4 a). As previously reported 31 , suboptimal vaccination with BA71ΔCD2 without HI-Ro or Frac-Ro conferred partial protection, with four out of six pigs (66.6%) surviving the lethal challenge, while all unvaccinated animals died between days 6–7 post-challenge (p.c.) (Fig. 4 b). Importantly, in line with the lower immune responses induced when using HI-Ro or Frac-Ro (Fig. 3 ), pigs vaccinated with these adjuvants showed a notable drop in the vaccine efficacy. Indeed, only 2 out of 6 pigs (33.3%) from each group inoculated with HI-Ro or Frac-Ro survived the challenge (Fig. 4 b). Moreover, only one of these four surviving pigs showed robust protection, while the other three pigs presented moderate to severe ASF clinical signs and temperatures exceeding 41ºC (Fig. 4 c-d). The decline in the efficacy of adjuvanted vaccinations was also reflected by an uncontrolled viremia. Indeed, only one surviving pig was negative until the end of the study (Fig. 5 a ) . Importantly, all pigs vaccinated with any of the two adjuvants, including the surviving ones, showed high levels of virus shedding in nasal swabs (Fig. 5 b ) . This uncontrolled virus replication was further validated analysing virus loads in lungs and gastrohepatic lymph nodes by qPCR, which were positive in all pigs vaccinated with adjuvant (Fig. 5 c-d). These results were in contrast with the protection afforded in not adjuvanted BA71ΔCD2 vaccinated pigs, which did not show high systemic or local virus loads (Fig. 5 a-c), demonstrating that HI-Ro or Frac-Ro hamper BA71ΔCD2-induced protective immunity. 4. Reduced BA71ΔCD2 vaccine efficacy mediated by HI-Ro and Frac-Ro is associated with a hampered replication capacity in stimulated macrophages. We have previously demonstrated that HI-Ro has anti-viral properties, reducing the replication capability of virulent ASFV and PRRSV strains in macrophages 32 . Thus, the drop in adjuvanted BA71ΔCD2 vaccine efficacy might result from the lower antigen doses generated due to the impaired LAV replication, which is not compensated by the enhanced functionality of BA71ΔCD2-infected macrophages upon HI-Ro or Frac-Ro treatment (Fig. 2 ). To explore this hypothesis, we measured in vitro the replication kinetics of BA71ΔCD2 in PAMs treated or untreated with HI-Ro or Frac-Ro. Percentages of infected cells were quantified by time-lapse analysis of macrophages infected with a BA71ΔCD2 expressing a fluorescent fusion protein of the late viral structural protein p54 (BA71ΔCD2-mWasabi). Importantly, the presence of either HI-Ro or Frac-Ro resulted in a significant decrease of the replication capacity of the vaccine strain, regardless of the initial virus dose used (Fig. 6 a-b; Supplementary Fig. 4a-b ). These results were further validated by flow cytometry at 48- and 72- hours post-infection, analysing cells positive for the late expressed viral protein p72. Indeed, both HI-Ro and Frac-Ro significantly decreased the percentage of infected cells (Fig. 6 c; Supplementary Fig. 4c ). This effect was probably mediated by the activation of macrophages, since similar results were obtained when infecting the cells together with stimulatory components IFN-γ or LPS (Fig. 6 c; Supplementary Fig. 4c ). Indeed, a short stimulation with HI-Ro or Frac-Ro prior to BA71ΔCD2 infection did not modify the replication capacity of the vaccine virus ( Supplementary Fig. 5 ), thus further suggesting that a full activation of macrophages is required for the anti-viral effect. Importantly, also the mean fluorescent intensity of p72 was decreased in BA71ΔCD2-infected and HI-Ro- or IFN-γ-treated cells, indicating the lower replicative capacity of the vaccine virus at the single cell level (Fig. 6 d; Supplementary Fig. 4d ). In this line, analysis of percentages of infected cells by the detection of the early viral protein p30 also demonstrated the lower replicative capacity of BA71ΔCD2 in the presence of HI-Ro or Frac-Ro (Fig. 7 ), denoting that the antiviral activity affects early steps of the virus replication cycle. Notably, these differences did not result from a cytotoxic effect of the treatments, since the percentages of alive cells were not significantly decreased in the presence of HI-Ro or Frac-Ro (Fig. 6 e; Supplementary Fig. 4e ). Discussion While waiting for the development of efficient subunit vaccines against ASF, the use of ASFV-based LAVs with improved biosafety profiles might represent a temporary solution in affected areas 10 . Here, we have shown that any attempt to increase LAVs biosafety by the use of bacterium-derived adjuvants should avoid the triggering of an antiviral state in macrophages. The use of immunostimulants such as HI-Ro and Frac-Ro might counteract the ASFV-mediated inhibition of macrophage functionality, and thus help in the induction of protective immune responses. However, inherent with this enhanced functional state of macrophages was the activation of an antiviral response, which in turn decreased intracellular LAV replication and thus the amount of antigens required to induce protective immunity. Therefore, the use of adjuvants combined with ASFV-based LAVs must target specific immune components and/or signalling pathways promoting innate immune responses, while allowing enough LAV replication to reach the threshold of viral antigen levels required to trigger a protective adaptive immunity. The use of LAVs is the only current vaccine strategy conferring solid protection against ASF, as evidenced by the two LAV ASF vaccines registered in Vietnam 7 . However, their implementation in the field is limited due to biosafety concerns 6 , 11 . Indeed, the balance between virus attenuation and immunogenicity is very subtle, with the consequent potential of reversion to virulence 5 , 8 , 33 . However, despite these concerns, there is an open discussion on the potential benefits derived from a rational use of LAVs in areas affected by virulent ASFV strains 10 . Thus, the optimization of LAVs to improve their biosafety profile may contribute to controlling ASFV spread while reducing the risk of adverse events. In this scenario, the use of adjuvants represents a potential strategy that deserves investigation. Adjuvants boost vaccine-induced immune responses while reducing the amount of antigen required for effective immunization 34 . Therefore, the combination of adjuvants with ASF LAVs might lead to a reduction of the vaccine dose with the subsequent drop in potential biosafety issues. However, vaccines based on live viruses have a robust endogenous stimulatory capability, and typically do not need adjuvants to trigger immune responses 25 . But this concept should be reconsidered for viruses suppressing innate immune signalling pathways to evade antiviral immunity. Indeed, here we have demonstrated that BA71ΔCD2 combined with HI-Ro or Frac-Ro rescues the functionality of infected macrophages, promoting cytokine production and the upregulation of activation markers. These results demonstrate that there is room for adjuvant-mediated enhancement of ASF LAV immunogenicity. The attenuation of ASFV is usually achieved by mutations or deletions in genes involved in immunosuppressive mechanisms 5 , 14 , such as the CD2v-encoding gene deleted from the BA71ΔCD2 35,36 . However, these genetic modifications in such a complex virus do not completely revert its capability to compromise macrophage functionality 37 – 39 . Thus, it is likely that similar results obtained with the BA71ΔCD2 would be obtained using other ASFV attenuated strains. Finally, this strategy might be also applied to LAVs against porcine reproductive and respiratory syndrome (PRRS) and classical swine fever (CSF) viruses, which also infect macrophages and suppress innate immunity 40 , 41 . The enhanced functionality of BA71ΔCD2-infected macrophages observed in vitro upon both HI-Ro and Frac-Ro stimulation did not result in an improvement in vaccine efficacy against a lethal challenge in vivo , regardless of the adjuvant used. Instead, the protection afforded upon administration of the adjuvanted LAVs was significantly reduced. Vaccine efficacy against any pathogen depends on several factors, but the antigen levels and the type and magnitude of the induced immune responses are particularly critical 42 . In this line, HI-Ro or Frac-Ro adjuvanted BA71ΔCD2 generated lower levels of ASFV-specific humoral and cellular responses than the non-adjuvanted LAV, but did not alter the quality of virus-specific immune responses. Indeed, the results indicate that HI-Ro and Frac-Ro neither significantly modified virus-specific antibody isotypes, nor the ratio between antibody and cellular responses induced in each pig. Thus, it is likely that the decreased vaccine efficacy was linked to the insufficient induction of virus-specific antibodies and T cells, which did not reach the required thresholds to protect the animals. Mechanistically, it is feasible to hypothesise that the lower immunogenicity of the adjuvanted LAV is associated with a reduced replication rate of the BA71ΔCD2 LAV in vivo , thus not producing the required antigen levels to induce a protective immunity. This hypothesis is supported by the lower capability of the BA71ΔCD2 to replicate in vitro in the presence of HI-Ro or Frac-Ro, likely as a consequence of the antiviral state induced in activated macrophages. Indeed, ASFV is sensitive to interferon-induced responses, as shown both in vitro 38 and in vivo 43 . Moreover, an elegant study in a mouse model showed that short-term IFN-I blockade during immunization with several viral vaccines enhances their efficacy through a transient “spike” in antigen levels 44 . Thus, the capability of Rothia -derived components to induce the expression of interferon stimulated genes (ISGs) in BA71ΔCD2-infected macrophages might restrain the virus replication rate with the consequent decrease of antigen production. However, we cannot discard other factors being also responsible for the decreased adjuvanted vaccine efficacy, since the events underlying vaccine-induced immunity in vivo are highly complex, with many cell subsets involved. This complexity is evidenced by studies performed using adjuvants for live attenuated influenza virus vaccines. In mice, a-C-GalCer increased influenza-specific humoral and cellular responses in a Natural killer T cell-dependent manner, and subsequently reduced both morbidity and mortality after challenge 29 . However, in line with our results, a-C-GalCer impaired the replication of the live vaccine in pigs, reducing vaccine-induced cross-protective immune responses 30 . Thus, further in vivo studies not restricted to LAV-macrophage interactions analyses in vitro are required to understand the adjuvant-triggered mechanisms directly modulating vaccine responses. The results discussed above clearly indicate that a fine-tuned and rational selection of adjuvants is necessary to exploit their use for ASF LAVs. Thus, to obtain an adjuvanted vaccine formulation with higher biosafety profiles, further research is needed to better characterize the innate immune responses associated with a favourable outcome upon ASFV or LAV infections. In this context, several early correlates of protection have been recently identified upon immunization with the incompletely attenuated ASFV strain Estonia 2014 45 . In this model, the prompt activation of antigen presenting cells (APC), plasma cells and T cells correlated with the induction of protective immunity. However, a systemic antiviral response and high levels of IFNa in serum were also identified as favourable parameters. These results contrast with our association of an antiviral response in macrophages with a decreased vaccine efficacy. However, this study analysed systemic responses in blood in a model characterised by long viremia, and infection outcomes are usually linked to early events occurring at the site of initial viral replication 46 – 48 . This is especially important for completely attenuated ASFV strains such as BA71ΔCD2 and other LAVs, which usually do not spread systemically or have short viraemic periods 5 . Thus, further studies are required to identify early and local correlates of protection induced by LAV, which will help to decipher the innate immune components that need to be targeted by adjuvants. Rationally selected adjuvants should achieve a good balance between an enhanced immune functionality in innate immune cells, and the capability of the vaccine virus to replicate and produce the required antigen levels to trigger a protective adaptive immune response. Methods Preparation of heat-inactivated R. nasimurium (HI-Ro) and cell-free supernatant (Frac-Ro). Growth of R. nasimurium to obtain the required multiplicity of infection (MOI) was performed as previously described 32 . The bacterium was quantified by serial dilutions and plating, and inactivated at 65°C for 1 hour. Inactivation was confirmed by absence of overnight growth on chocolate agar at 37°C with 5% of CO 2 . To obtain the cell-free supernatant, a plate with a R. nasimurium overnight culture was washed using 10 mL of phosphate-buffered saline (PBS), and centrifuged at 2.500 x g 10 min. The cell-free resulting supernatant was sterilized using a 0.2 µm filter. The different fractions from the supernatant were obtained using centrifugal filters with a molecular weight cut-off (MWCO) of 100 or 10 kDa (Amicon UFC9100, UFC9010). Supernatant fractions of ≥ 100, 100 − 10, ≤ 10 MWCO kDa were recovered with 10 mL of Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco). For in vitro stimulatory assays, HI-Ro was first centrifuged at 2500 x g 10 min and resuspended in the corresponding media at MOI 50. Frac-Ro was diluted 1:2 in the corresponding x2 concentrated media. For in vivo experiments, HI-Ro was used as 10 7 cfu/mL and Frac-Ro was diluted 1:2 with PBS. Viruses The virulent ASFV strain Georgia2007/1 (genotype II) was kindly provided by Dr. Linda Dixon (WOAH reference laboratory, Pirbright Institute, UK), growth in PAMs, and titrated by hemadsorption assay 49 . The BA71ΔCD2 is a live attenuated strain lacking the CD2v gene (EP402R) obtained by homologous recombination from the parental virulent BA71 strain 50 . It was grown in the stabilised COS-1 cell line (ATCC), and titrated by immunoperoxidase monolayer assay (IPMA) 50 . Titres were calculated by the Reed and Muench method, and expressed as 50% haemagglutination activity units (HAU 50 )/mL or 50% tissue culture infectious dose. TCID 50 titres obtained from IPMA were converted to plaque forming units (pfu) applying the Poisson distribution (pfu/mL ≈ 0.7 × TCID 50 /mL). Porcine alveolar macrophages and cytokine quantification from cell culture supernatants Porcine alveolar macrophages (PAMs) were obtained through lung lavage of healthy animals (from Landrace, Landrace x Duroc and Landrace x Large White pig breeds) as previously described 32 . Cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen), 1% of penicillin-streptomycin/mL (P/S) (Invitrogen), 1% of L-glutamine (Invitrogen), and 0.5% of nystatin (Invitrogen). For in vitro stimulation assays, three to four lots of PAMs were seeded in 96-well flat-bottom plates at 5-6x10 5 cells/well and left overnight at 37°C. Then, cells were incubated with alive R. nasimurium , HI-Ro, or Frac-Ro at the indicated time points. After stimulation, supernatants were collected and stored at -80°C for TNF (R&D system), IL-1β (R&D system), and IFN-γ (King Fisher) quantification by ELISA, following the manufacturing procedure. RNA-seq library preparation and sequencing Four lots of PAMs were seeded at 5x10 6 cells/well in a 6-well flat-bottom plate, and left overnight at 37°C with 5% of CO 2 . PAMs were stimulated for 6 or 24 hours with Frac-Ro (dilution 1/2). Non-stimulated cells were used as control. Total RNA was isolated using the RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. To ensure RNA quality, DNase I treatment was performed for 15 min at room temperature. RNA was sequenced at the Centre Nacional d’Anàlisi Genòmica (CNAG), Barcelona, Spain. Total RNA concentration was quantified using Qubit RNA BR Assay kit (ThermoFisher Scientific) and the RNA integrity was estimated by Agilent Bioanalyzer. The RNASeq libraries were prepared with KAPA mRNA HyperPrep Kit (Roche) following the manufacturer’s recommendations starting with 500 ng of total RNA as the input material. The library was quality controlled on an Agilent 2100 Bioanalyzer with the DNA 7500 assay. The libraries were sequenced on NovaSeq 6000 (Illumina) with a read length of 2x51bp, following the manufacturer’s protocol for dual indexing. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis. RNA-seq bioinformatic analysis Illumina reads were aligned to the Sus scrofa reference genome (Sscrofa11.1) with STAR (v2.7.8a) using ENCODE-recommended parameters 51 . Gene-level quantification used RSEM (v1.3.0) against Ensembl Sus scrofa annotation release 110 52 . Differential expression was tested with limma (v3.42.3). TMM was applied to compute library-size/compositional scaling factors, and voom converted counts to log2-counts-per-million (logCPM) while modeling the mean–variance trend on logCPM to derive observation-level precision weights 53 , 54 . Linear models were fit to the weighted logCPM values. Given the paired design, inter-individual effects were modeled with duplicateCorrelation; we extracted pairwise contrasts and the treatment × vaccination interaction. Genes were called DE at Benjamini–Hochberg FDR < 0.05. Functional enrichment used DAVID ( http://david.ncifcrf.gov/ ) 55 . Animal experiment Landrace x Large White breed males were subjected to a seven-day acclimation period prior to vaccination at seven weeks of age. Animals were fed ad libitum . Pigs were divided into four groups of six animals. Three different vaccine formulations were tested: non-adjuvanted vaccine, HI-Ro-adjuvanted vaccine [10 7 cfu/mL], and Frac-Ro-adjuvanted vaccine [dilution 1:2]. In all cases, vaccinated animals were intranasally inoculated with 10 4 pfu/animal of the attenuated LAV prototype BA71ΔCD2. Each animal received 2 mL of the vaccine diluted in PBS via intranasal inoculation (1 mL/nostril). The remaining six animals were used as unvaccinated controls and received 2 mL of PBS alone (1 mL/nostril). At day 22 post-vaccination (p.v.), all animals received 2 mL of an intranasal challenge containing 10 5 HAU 50 of the highly virulent ASFV strain Georgia2007/1 (1 mL/nostril). Rectal temperature and clinical signs were monitored daily during all the experiments as previously described 56 . Sera and nasal swabs were taken at 0, 7, 15, and 22 days p.v., and 0, 7, 13, and 20 days post-challenge (p.c.) to quantify viral loads by qPCR and ASFV-specific antibodies by ELISA. PBMCs were isolated at day 22 p.v. (day 0 p.c.) to measure ASFV-specific IFN-γ-producing cells by ELISpot. Finally, on the last day of the study lung and gastrohepatic lymph node tissues from each animal were isolated and kept at -80 until analysis. Quantitative PCR for the detection of ASFV ASFV loads in sera, nasal swabs, and tissues were assessed by SYBR Green qPCR targeting the ASFV serine protein kinase gene (R298L; PK) as previously described 50 . Briefly, the viral genomic DNA was obtained using IndiMag® Pathogen Kit (INDICAL Bioscience) in a semi-automated manner by using a KingFisher System (Thermo) according to the manufacturer’s instructions. qPCR amplifications were performed in duplicates using the corresponding standards for absolute quantification. The results were expressed as log 10 genome-equivalent copies (GEC) per millilitre of sera or nasal swab, or per 0.1 g of tissue. The detection limit of the technique was 10 3 GEC/mL. Enzyme-linked immunosorbent assay (ELISA) ASFV-specific antibodies in pig sera were detected by the WOAH-approved ELISA based on soluble extracts from ASFV-infected cells 57 . Samples were serially diluted from 1/100 to 1/1.562.500 to calculate the endpoint-titration, with a cut-off defined as the average negative control plus three times the standard deviation of the negative control. Positive sera were detected using the secondary peroxidase-conjugate antibodies: rabbit anti-pig IgG at 1/20000 dilution (Sigma-Aldrich), anti-pig IgG1 at 1/1000 (Bio-Rad), anti-pig IgG2 at 1/1000 (Bio-Rad), or anti-pig IgM at 1/100000 (BioRad). Soluble 3,3’,5,5’-tetramethylbenzidine (TMB, Sigma-Aldrich) was used as a specific peroxidase substrate. H2SO4 at 1N was used as a stop solution and plates were read at 450nm. All samples were run in technical duplicates. IFN-γ enzyme-linked immunosorbent spot (ELISpot) assay The IFN-γ-ELISpot assay was performed as previously described 58 . Briefly, IFN-γ-secreting cells in PBMCs were measured by ELISpot assay using the purified mouse anti-pig IFN-g (clone P2G10, BD Pharmingen) as capture antibody and biotinylated mouse anti-porcine IFN-g antibody (clone P2C11, BD Pharmingen) as detection antibody. PBMCs were stimulated with BA71ΔCD2 or Georgia2007/1 at a MOI of 0.2, and incubated for 16 hours at 37˚C, 5% CO2. Sample scoring ≥ 300 spots/million PBMCs received a score of 300. Samples approaching this ceiling were not diluted further, so values at the cap may underestimate true spot counts. Luminex-based multiplex assay PAMs were stimulated for 24 hours with the three cell-free supernatant fractions and supernatants were recovered and kept at -80ºC until analysis. Cytokine levels were quantified using the Luminex xMAP technology following the manufacturer’s instructions. Measurements included IFN-α, IL-1b, IL-4, IL-6, IL-12, and IL-10, IL-12p40 (ProcartaPlex Porcine Cytokine & Chemokine Panel 1; Thermo-Fisher Scientific). Concentrations of each cytokine were calculated using the xPONENT software (Luminex). Microfluidic quantitative PCR assay Five lots of PAMs were seeded at 6x10 5 cells/well in a 48-well flat-bottom plate and left overnight at 37°C with 5% of CO 2 . Cells were infected during 2h with the BA71ΔCD2 ASFV strain at MOI 0.1 in 100 µl/well of RPMI supplemented with 1% L-glutamine. After infection, cells were stimulated overnight with HI-Ro or Frac-Ro. Total RNA was isolated using the RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. Concentration was quantified using Qubit RNA BR Assay kit (ThermoFisher Scientific) and the RNA integrity was estimated by Agilent Bioanalyzer. cDNA was obtained from 150 ng of total RNA using the PrimeScript RT reagent Kit (Takara, Japan) following the manufacturer’s instructions. Primer design ( Supplementary Data 2 ) and validation was performed as previously described 59 . Gene expression levels were measured in duplicates using a microfluidic qPCR with the 48.48 Dynamic Array integrated fluidic circuit of the Biomark HD system (Fluidigm Corporation). Data was analyzed applying the relative standard curve method and using the Fluidigm Real-Time PCR analysis software 4.1.3 and the DAG expression software 1.0.5.6 60 . Target gene expression levels were normalized against the average of three reference control genes (YWHAZ, RPL4 and GAPDH), and z-score normalized values were represented in a Heatmap. Time-lapse microscopy of viral infection kinetics The BA71ΔCD2-mWasabi fluorescent virus, encoding the mWasabi fluorescent protein ( https://www.fpbase.org/protein/mwasabi/ ) fused to the C-terminus of the p54 ASFV protein, was generated by CRISPR technology as previously described 32 . To evaluate the capability of BA71ΔCD2 to replicate in the presence of HI-Ro and Frac-Ro, PAMs were seeded at 1.5x10 5 cells/well and infected with the BA71ΔCD2-mWasabi at MOI 0.1 or 3.5 in RPMI supplemented with 1% L-glutamine for 2 hours. Next, cells were treated with HI-Ro or Frac-Ro. Viral infection kinetics were then quantified over 72 hours by time-lapse microscopy using an IncuCyte® SX5 (Sartorius BioAnalytical Instruments Inc, CA, USA). Specifically, plates were imaged every 2h at 20x (4 fields of view per well) using the device’s “AI Scan” module in two channels (phase and green) using default acquisition parameters (green acquisition time 300 ms). Cell detection was performed using the “AI Cell Health” module (Segmentation Sensitivity = 0.6) without filtering for cell size. Classification of detected cells across the dimensions “uninfected/infected” was finally performed using the “Cell-by-Cell Classification” module: infected cells were identified based on a Green Mean Intensity threshold of 0.6 GCU (set empirically to have ~ 99% of cells in uninfected controls shown as negative). Flow cytometry PAMs were seeded at 6x10 5 cells/well in a 48-well flat-bottom plate infected with BA71ΔCD2 at MOI 0.1 for 2 hours in 50 µl/well of RPMI supplemented with 1% L-glutamine for. Next, cells were treated with HI-Ro or Frac-Ro. At 48 h post-infection, cells were stained for viability with LIVE/DEAD Fixable Violet Dead Cell Stain Kit following the manufacturer’s instructions (ThermoFisher Scientific). Blockage of Fc receptors was performed with PBS containing 5% of porcine serum (Gibco) for 15 min on ice prior to antibody staining. For intracellular staining of virus-infected PAMs, cells were fixed and permeabilized with the BD Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer’s protocol, and incubated during 30 min on ice in Perm/Wash buffer with anti-p72 antibody (Eurofins Ingenasa; M.11.PPA.I1BC11) or anti-p30 antibody (hybridoma 1E12F5H2) kindly provided by M. Domínguez for ASFV. Then, cells were incubated with the secondary antibodies anti-mouse IgG1 (eBioscience; 25-4015-82) and anti-mouse IgG2a (Thermo fisher; A-21134), respectively. In all cases, samples were acquired in a BD FACSAria IIu flow cytometer (BD Biosciences) and data was analysed using FlowJo v10.8.1 software (Tree Star Inc). Statistical analyses Graphics were created and analysed using the Prism version 8.0.2 software (GraphPad), and RStudio. Each statistic test is indicated in the corresponding figure legend. Statistical difference was set up at: ns p > 0.05; *p \(\:\le\:\) 0.05; **p \(\:\le\:\) 0.01; ***p \(\:\le\:\) 0.001; ****p \(\:\le\:\) 0.0001. Ethics statement Animal care and procedures were conducted following the guidelines of the Good Experimental Practice and with the acceptance of the Ethics Committee on Animal Experimentation of the Generalitat de Catalunya (protocol code: 12121, approved on 2024/10/09). All experiments were performed in the biosafety level 3 facilities at Centre de Recerca en Sanitat Animal (IRTA-CReSA, Barcelona). Declarations Competing interest: The authors declare no conflicts of interest. Funding This research was funded by the Spanish Ministry of Science and Innovation, MICIU/AEI/ 10.13039/501100011033 , grant PID2022-136312OB-I00 (F.R. and J.A.). Author Contribution Author Contributions: Designed the research: F.R. and J.A. Performed the research: A.T-M., S.M-T., D.M-M., J.M-B, Y.Z., M.J.N., M.M., P.M., J.G-O., B.M-M., E.V., A.C. Analysed and interpreted data: J.A., F.R., E.G-F., S.P., F.A., V.A., A.E-C. Wrote the manuscript: J.A., A.T-M. Reviewed the manuscript: J.A., A.E-C., F.R. All authors have approved the submitted version. Acknowledgement We thank Animal Facility and Clinical and preclinical studies units from IRTA-CReSA for their technical support. Data Availability Sequence data that support the findings of this study have been deposited in the Gene Expression Omnibus repository: https://www.ncbi.nlm.nih.gov/geo/, code GSE288520. References Portugal, R. et al. Six adenoviral vectored African swine fever virus genes protect against fatal disease caused by genotype I challenge. J. Virol. 98, e0062224 (2024). Liu, W. et al. A new vaccination regimen using adenovirus-vectored vaccine confers effective protection against African swine fever virus in swine. Emerg. Microbes Infect. 12, 2233643 (2023). Rock, D. L. 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supernatant (Whole SN), three different fractions of the supernatant separated by molecular sizes (Frac-Ro\u0026gt;100 kDa, Frac 100-10 kDa, Frac\u0026lt;10 kDa), or heat-inactivated \u003cem\u003eR. nasimurium\u003c/em\u003e(HI-Ro) at the indicated multiplicity of infection (MOI). Levels of IFN-g (\u003cstrong\u003ea\u003c/strong\u003e) and TNF (\u003cstrong\u003eb\u003c/strong\u003e) in cell supernatants were measured by ELISA. (\u003cstrong\u003ec-d\u003c/strong\u003e) PAMs were stimulated for 6 (\u003cstrong\u003ec\u003c/strong\u003e) or 24 (\u003cstrong\u003ec-d\u003c/strong\u003e) hours with the fraction of the supernatant containing components with a molecular weight \u0026gt; 100 kDa (Frac-Ro). (\u003cstrong\u003ec\u003c/strong\u003e) List of representative Gene Ontology (GO) terms enriched among differentially expressed (DE) genes. (\u003cstrong\u003ed\u003c/strong\u003e) Heatmap depicting the z-score normalized log2CPM values from representative DE genes in the indicated categories. Non-stimulated cells were used as reference. (\u003cstrong\u003ea-b\u003c/strong\u003e) Significant differences were determined using a one-way ANOVA (* p-value ≤ 0.05).\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7815447/v1/a7f93c226b04b1f7445fd495.jpeg"},{"id":95327536,"identity":"44be0504-bcfd-492b-8abe-2162e10755e7","added_by":"auto","created_at":"2025-11-06 18:17:41","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":209351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHI-Ro and Frac-Ro boost the functionality of BA71ΔCD2-infected macrophages\u003c/strong\u003e. (\u003cstrong\u003ea-d\u003c/strong\u003e) Porcine alveolar macrophages (PAMs) were infected with the vaccine prototype BA71ΔCD2 for 2 hours (MOI 0.1), and then treated with HI-Ro (MOI 50), Frac-Ro, IFN-g (0.1 mg/mL), LPS (10 mg/mL), or left untreated. (\u003cstrong\u003ea-c\u003c/strong\u003e) At 72h post-infection levels of TNF (\u003cstrong\u003ea\u003c/strong\u003e), IFN-g (\u003cstrong\u003eb\u003c/strong\u003e), and IL-1b (\u003cstrong\u003ec\u003c/strong\u003e) were quantified by ELISA in supernatants. Non-infected cells were used as control. (\u003cstrong\u003ed\u003c/strong\u003e) Expression levels of 32 genes representative of innate immune responses were quantified at 24 hours post-infection by microfluidic quantitative PCR assay. The heatmap shows z-score normalized gene expression. Significant differences were determined using one-way ANOVA (\u003cstrong\u003ea-c\u003c/strong\u003e) or t-test comparing infected and non-infected PAMs (\u003cstrong\u003ed\u003c/strong\u003e) (* p-value ≤ 0.05, **≤ 0.01, ***≤ 0.001, and ****≤ 0.0001).\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7815447/v1/2f44a5208352b97f57c9a355.jpeg"},{"id":95327541,"identity":"39430747-6d11-4e2a-884d-bb5a0325823d","added_by":"auto","created_at":"2025-11-06 18:17:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":251053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination of intranasal BA71ΔCD2 vaccination with HI-Ro or Frac-Ro impairs the induction of ASFV-specific cellular and humoral responses.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Schematic representation of the experimental design. (\u003cstrong\u003eb\u003c/strong\u003e) IFN-g-producing cells in peripheral blood mononuclear cells (PBMCs) obtained at day 22 post-vaccination were quantified by ELISpot. Cells were stimulated \u003cem\u003ein vitro\u003c/em\u003e with the ASFV-strains BA71ΔCD2 or Georgia2007/1. (\u003cstrong\u003ec-e\u003c/strong\u003e) ASFV-specific IgG (\u003cstrong\u003ec\u003c/strong\u003e), IgG1, IgG2 (\u003cstrong\u003ed\u003c/strong\u003e), and IgM (\u003cstrong\u003ee\u003c/strong\u003e) in serum at the indicated time-points were titrated by ELISA. (\u003cstrong\u003ef\u003c/strong\u003e) Spearman correlation analysis between the number of ASFV-specific IFN-g-producing cells and the ASFV-specific IgG titre for each animal. Significant differences were determined using one-way ANOVA (\u003cstrong\u003eb-e\u003c/strong\u003e) or Spearman correlation (\u003cstrong\u003ef\u003c/strong\u003e) (* p-value ≤ 0.05, ****≤ 0.0001).\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7815447/v1/911d168a30a6ccd79d0b3fea.jpeg"},{"id":95523829,"identity":"11dc2901-8c76-4e16-88c7-464d2c773c2a","added_by":"auto","created_at":"2025-11-10 10:01:07","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":304735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBA71ΔCD2 efficacy against a lethal ASFV challenge is reduced when combined with HI-Ro or Frac-Ro. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eGraphical representation of the experimental design. (\u003cstrong\u003eb\u003c/strong\u003e) Survival plot showing the percentage of alive pigs at the indicated time points after intranasal challenge with Georgia2007/1. The number of surviving pigs at the end of the experiment is indicated in brackets. (\u003cstrong\u003ec-d\u003c/strong\u003e) Daily clinical scores (\u003cstrong\u003ec\u003c/strong\u003e) and rectal temperatures (\u003cstrong\u003ed\u003c/strong\u003e) of individual pigs after intranasal challenge. The severity of the clinical signs (score value) is represented by a coloured gradient: green (absence), yellow (mild), and orange/red (severe).\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7815447/v1/9f8a82896c51de5829525b99.jpeg"},{"id":95327542,"identity":"4cff14cc-e4ac-4c5f-82b2-7185f462868c","added_by":"auto","created_at":"2025-11-06 18:17:41","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":321551,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePigs vaccinated with BA71ΔCD2 without adjuvants\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eshow lower viremia and virus shedding after lethal challenge\u003c/strong\u003e. (\u003cstrong\u003ea-b\u003c/strong\u003e) Virus loads in blood serum (\u003cstrong\u003ea\u003c/strong\u003e) and nasal swabs (\u003cstrong\u003eb\u003c/strong\u003e) at days 0, 7, 13, and 20 post-challenge measured by qPCR. Genomic equivalent copies (GEC) were quantified by the detection of the ASFV gene \u003cem\u003ePK\u003c/em\u003e. (\u003cstrong\u003ec-d\u003c/strong\u003e) Virus loads in lung (\u003cstrong\u003ec\u003c/strong\u003e) and gastrohepatic lymph node (\u003cstrong\u003ed\u003c/strong\u003e) at the day of sacrifice of each animal. Black Filled symbols represent animals surviving until the end of the study, and white symbols represent animals sacrificed before the end of the study. Significant differences were determined using one-way ANOVA (* p-value ≤ 0.05).\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7815447/v1/7598dfb8e5ee829c1c486826.jpeg"},{"id":95524756,"identity":"b0234519-35f8-4ecd-9e88-591ae8e6d415","added_by":"auto","created_at":"2025-11-10 10:03:26","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":215567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBA71ΔCD2 replication capability is reduced when combined with HI-Ro and Frac-Ro. \u003c/strong\u003e(\u003cstrong\u003ea-b\u003c/strong\u003e) Porcine alveolar macrophages were infected with the fluorescently labelled BA71ΔCD2-mWasabi at MOI 0.1. Two hours post-infection, cells were treated with HI-Ro (MOI 50), Frac-Ro or left untreated. (\u003cstrong\u003ea\u003c/strong\u003e) The percentages of BA71ΔCD2-infected cells were analysed every 2 hours using time-lapse Incucyte analysis. (\u003cstrong\u003eb\u003c/strong\u003e) Area under the curve (AUC) for each condition obtained from the kinetics of percentages of infected cells. (\u003cstrong\u003ec-e\u003c/strong\u003e) BA71ΔCD2-infected PAMs (MOI 0.1) treated with HI-Ro (MOI 50), Frac-Ro, IFN-g (0.1 mg/mL), LPS (10 mg/mL) or left untreated were analysed at 72 h post-infection by flow cytometry. The percentages of BA71ΔCD2-infected cells (\u003cstrong\u003ec\u003c/strong\u003e), the mean fluorescent intensity (MFI) of the p72 ASFV protein (\u003cstrong\u003ed\u003c/strong\u003e), and the percentage of alive cells (\u003cstrong\u003ee\u003c/strong\u003e) are shown. Significant differences were determined using a one-way ANOVA (* p-values ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001).\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7815447/v1/5721d9e9bb189f4b547c29da.jpeg"},{"id":95327549,"identity":"b0ecac56-9512-4755-a856-e8c03dad7343","added_by":"auto","created_at":"2025-11-06 18:17:41","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":197234,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHI-Ro and Frac-Ro impair the early expression of the ASFV protein p30.\u003c/strong\u003e (\u003cstrong\u003ea-d\u003c/strong\u003e) Porcine alveolar macrophages were infected with the BA71ΔCD2, and 2 hours post-infection were treated with HI-Ro (MOI 50), Frac-Ro, or left untreated. At 24 (\u003cstrong\u003ea-b\u003c/strong\u003e) and 48 (\u003cstrong\u003ec-d\u003c/strong\u003e) hours post-infection, the percentages of p30-positive infected cells (\u003cstrong\u003ea, c\u003c/strong\u003e) and the MFI of the early expressed ASFV protein p30 (\u003cstrong\u003eb, d\u003c/strong\u003e) were measured by flow cytometry. Significant differences were determined using a one-way ANOVA (p-values of * p-values ≤ 0.05, ** ≤ 0.01).\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7815447/v1/7dcf3030515d0b9ca43247d9.jpeg"},{"id":107928184,"identity":"284db75b-7116-48ae-a9fa-0ed8ec19f48f","added_by":"auto","created_at":"2026-04-27 16:08:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2154861,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7815447/v1/a7cc4c7c-b24c-4af8-a901-9ff14da6568b.pdf"},{"id":95327534,"identity":"957e4f84-31d5-44bf-8d2c-799bca99b280","added_by":"auto","created_at":"2025-11-06 18:17:41","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14208,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7815447/v1/9c9df40ae2724da85d181258.xlsx"},{"id":95524436,"identity":"78ddcf74-e0fd-49dd-9fa9-9e2ee303155d","added_by":"auto","created_at":"2025-11-10 10:02:46","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13723,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7815447/v1/d600a7edcf26c0b698001add.xlsx"},{"id":95327552,"identity":"8dbefddc-6180-4350-bd64-a23072c30abb","added_by":"auto","created_at":"2025-11-06 18:17:41","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1295877,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7815447/v1/57c8ebafb2327554a1f249e0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Adjuvant-induced macrophage activation compromises BA71ΔCD2-mediated protection against African swine fever virus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe development of a safe and effective African swine fever (ASF) vaccine remains a major global challenge. The disease is one of the biggest threats affecting the pig industry worldwide, with a complex epidemiological pattern in Africa, and currently spreading in Asia and Europe through the infection of domestic pigs and wild boars (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://efsa.onlinelibrary.wiley.com/doi/\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2903/j.efsa.2023.8016\u003c/span\u003e\u003cspan address=\"10.2903/j.efsa.2023.8016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e While the development of subunit vaccines is progressing\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, it continues facing important limitations due to the lack of knowledge on protective antigens and immune responses\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In contrast, several experimental live attenuated vaccines (LAVs) confer robust immunity and protection against lethal challenge\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, biosafety concerns hold back their implementation in the field\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Indeed, two LAVs were approved in Vietnam in 2022\u003csup\u003e7\u003c/sup\u003e, but their widespread usage is not yet a reality due to the potential of reversion to virulence and the emergence of new recombinant strains\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Thus, while waiting for the generation of safe and effective subunit vaccines, the development of novel LAV-based strategies with enhanced biosafety profiles might boost their application in ASF endemic areas.\u003c/p\u003e\u003cp\u003eThe main target of ASF virus (ASFV) are monocytes/macrophages. With more than 150\u0026ndash;200 encoded proteins\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, the virus has a high capability to hijack the infected cells, modulating their functional responses and eventually causing fatal cytopathic effects\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Indeed, throughout its replication cycle, ASFV modulates cytokine production, antigen presentation pathways and apoptosis- or pyroptosis-programmed cell death in infected cells\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Consequently, macrophages are disabled to act as antigen presenting cells (APC), not providing to other immune cells the required signals for a proper induction of a protective adaptive immune response. To note, many LAVs or naturally attenuated ASFV strains inducing protective immunity trigger higher innate immune responses in infected macrophages than the parental virulent strains\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. This indicates that protective immunity induced by live ASFV-based immunisation is associated with the preservation of some degree of functionality in infected macrophages. However, the balance between virus attenuation and immunogenicity is subtle, and the generation of LAVs achieving a trade-off between biosafety and efficacy is complex. Notably, attempts to obtain safer LAVs through multiple gene deletion quite often result in reduced immunogenicity and loss of protective immunity\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe use of adjuvants has usually been restricted to subunit or inactivated vaccines, which commonly have a weak intrinsic capability to stimulate immune responses\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In contrast, LAVs have an inherent ability to activate the innate immunity upon their recognition by pattern recognition receptors (PRRs), thus promoting the induction of vaccine-specific adaptive immune responses\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, a few experimental studies have evaluated the co-administration of LAVs with adjuvants to test their ability to improve vaccine efficacy. In some cases, the combination of adjuvants with live vaccines has shown positive results, such as reduced vaccine side effects following vaccination with live \u003cem\u003eLeishmania major\u003c/em\u003e combined with CpG DNA\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and sterilising immunity against pathogenic simian human immunodeficiency virus (SHIV) in macaques vaccinated with a live attenuated SHIV expressing the adjuvant Ag85B\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, further research is required to evaluate the potential use of adjuvants for LAVs, since the effects will be highly adjuvant- and context-dependent. For instance, while the adjuvant a-C-galactosylceramide improved immune responses and protection in mice vaccinated with live attenuated influenza virus\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, another study in pigs showed that the closely related adjuvant a-galactosylceramide interfered with heterotypic cross-protective immune responses\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHere, we hypothesised that adjuvant-mediated activation of macrophages during ASF LAV immunisation would enhance vaccine efficacy, allowing for the reduction of the vaccine dose required to induce a protective immunity, and consequently improving its biosafety. To test this, pigs were intranasally immunised with a suboptimal dose of the ASF BA71ΔCD2 vaccine prototype, which confers partial protection against a lethal challenge\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. As adjuvants, we used two different formulations derived from \u003cem\u003eRothia nasimurium\u003c/em\u003e, a bacterium with immunostimulatory properties that robustly activates porcine alveolar macrophages\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The results demonstrated that despite the enhanced responsiveness of macrophages induced by these adjuvants, their administration with the BA71ΔCD2 did not improve vaccine immunogenicity and efficacy \u003cem\u003ein vivo\u003c/em\u003e. Indeed, the use of the adjuvants impaired vaccine-induced humoral and cellular responses, and the degree of protection against a challenge with a virulent ASFV strain. Importantly, further \u003cem\u003ein vitro\u003c/em\u003e analyses showed a lower replication capacity of BA71ΔCD2 in adjuvant-activated macrophages, indicating that the decreased vaccine effectiveness might result from a reduction in the antigen levels available to induce a protective immunity. These results represent a step forward in the understanding of early processes underlying LAV-induced immune protection, which might eventually help to rationally improve their biosafety profile.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003e1.\u003c/b\u003e \u003cb\u003eRothia nasimurium\u003c/b\u003e\u003cb\u003e-derived immunostimulants overcome the lack of responsiveness of BA71ΔCD2-infected macrophages\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eWe have recently characterized the immunostimulatory properties of heat-inactivated \u003cem\u003eRothia nasimurium\u003c/em\u003e (HI-Ro), demonstrating its capability to activate antigen presenting cells, thus showing its potential as a vaccine adjuvant\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Here, aiming to obtain a more purified product, we investigated whether the immunostimulatory components were present in cell-free supernatants from \u003cem\u003eR. nasimurium\u003c/em\u003e culture. Indeed, stimulation of porcine alveolar macrophages (PAMs) with supernatants induced IFN-γ, TNF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c), IL-12, and IL-1β (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e) secretion. Notably, the stimulatory capacity was only observed in a fraction of the supernatant containing components with a molecular weight\u0026thinsp;\u0026gt;\u0026thinsp;100kDa (Frac-Ro), and not in fractions containing smaller components (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c \u003cb\u003eand Supplementary Fig.\u0026nbsp;1\u003c/b\u003e). Interestingly, in contrast to Frac-Ro, HI-Ro did not stimulate IFN-γ production in macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), while both products induced robust TNF production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The stimulatory capacity of Frac-Ro was further demonstrated by analysing the transcriptome of treated macrophages by RNA-sequencing. Gene Ontology (GO) analysis of differentially expressed genes showed an enrichment in terms related to innate immunity, such as NF-κβ, MAPK, TLR and C-type lectin signalling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Despite the differences mentioned above regarding IFN-γ production, this transcriptomic signature was very similar to HI-Ro- and LPS-treated macrophages\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, upregulating genes encoding for cytokines and chemokines, factors involved in inflammatory pathways, activation markers and receptors, and interferon stimulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we evaluated whether HI-Ro or Frac-Ro treatment of macrophages infected with the ASFV vaccine prototype BA71ΔCD2 can overcome the viral induced immunosuppressive status, thus enhancing their functionality as antigen presenting cells. BA71ΔCD2-infected PAMs were stimulated with HI-Ro or Frac-Ro, and compared to IFN-γ or LPS, and their responsiveness was analysed by cytokine production and transcriptomic analysis. As previously demonstrated in macrophages infected with the highly virulent ASFV strain Georgia2007/1\u003csup\u003e32\u003c/sup\u003e, BA71ΔCD2 infection likewise failed to induce cytokine production in PAMs. However, this lack of responsiveness was overcome in HI-Ro and LPS-treated cells, which robustly secreted TNF. Stimulation with Frac-Ro and IFN-γ showed a similar but not statistically significant tendency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In contrast, IFN-γ production was only induced in BA71ΔCD2-infected cells following Frac-Ro stimulation, showing similar results with IFN-γ, but not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). To note, IL-1β was secreted only in HI-Ro- and LPS-treated macrophages although this effect did not reach statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Finally, the expression levels of 24 genes related to innate immunity were upregulated upon HI-Ro-stimulation of BA71ΔCD2-infected PAMs compared with non-treated and infected or non-infected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD; \u003cb\u003eSupplementary Data 1\u003c/b\u003e). In contrast, Frac-Ro only significantly increased the expression of a few genes such as \u003cem\u003eCCL3\u003c/em\u003e, \u003cem\u003eCCL4\u003c/em\u003e and \u003cem\u003eTNF\u003c/em\u003e. Taken together, these results indicate that the immunosuppressive state induced in ASFV-infected macrophages can be at least partially rescued by macrophage-activating components from \u003cem\u003eR. nasimurium\u003c/em\u003e, suggesting their potential as adjuvants for ASFV-based LAVs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e2. The use of HI-Ro or Frac-Ro as adjuvants impairs BA71ΔCD2-induced immunogenicity in pigs.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIntranasal vaccination with BA71ΔCD2 confers total or partial protection against a lethal challenge depending on the vaccine dose (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/36350837/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/36350837/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Thus, to evaluate the adjuvant potential of HI-Ro or Frac-Ro, we combined each of them with a suboptimal single dose of BA71ΔCD2 (10\u003csup\u003e4\u003c/sup\u003e pfu) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The use of adjuvants did not modify the safety profile of the BA71ΔCD2 vaccine prototype. Indeed, all vaccinated pigs showed lack of clinical signs, regardless of the use of adjuvants, with rectal temperatures below 41\u0026ordm;C and low clinical scores as in unvaccinated pigs (\u003cb\u003eSupplementary Fig.\u0026nbsp;2a-b\u003c/b\u003e). Consistently, virus loads in serum and nasal swabs were negative except for one animal vaccinated without adjuvant showing low levels of virus (\u003cb\u003eSupplementary Fig.\u0026nbsp;2c-d\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTo evaluate the effect of using HI-Ro or Frac-Ro as adjuvants, we next analysed cellular and humoral ASFV-specific immune responses induced post-vaccination (p.v.) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Importantly, although between-group differences did not reach statistical significance, point estimates for IFN-γ-producing cells and IgG titres at day 22 p.v. were lower with HI-Ro or Frac-Ro (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-c; \u003cb\u003eSupplementary Fig.\u0026nbsp;3a\u003c/b\u003e). Indeed, both ASFV-specific IgG1 and IgG2 antibody titres (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed; \u003cb\u003eSupplementary Fig.\u0026nbsp;3b-c\u003c/b\u003e), as well as ASFV-specific IgM titres at 14 days p.v. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee; \u003cb\u003eSupplementary Fig.\u0026nbsp;3d\u003c/b\u003e), were lower in the two groups vaccinated with adjuvants. Finally, a correlation analysis between ASFV-specific IFN-γ-producing cells and IgG titres at day 22 p.v. demonstrated that pigs responding to vaccination concomitantly triggered both cellular and humoral responses, regardless of the vaccination group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Overall, these results indicate that the use of HI-Ro or Frac-Ro as BA71ΔCD2 adjuvants dampens vaccine-induced immunity, equally affecting both systemic cellular and humoral responses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3. Combination of BA71ΔCD2 with HI-Ro or Frac-Ro reduces vaccine efficacy against a lethal ASFV infection.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the protection induced by BA71ΔCD2 combined with HI-Ro or Frac-Ro, we next challenged the pigs at 22 days p.v. with a lethal intranasal inoculation of the virulent Georgia2007/1 strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). As previously reported\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, suboptimal vaccination with BA71ΔCD2 without HI-Ro or Frac-Ro conferred partial protection, with four out of six pigs (66.6%) surviving the lethal challenge, while all unvaccinated animals died between days 6\u0026ndash;7 post-challenge (p.c.) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Importantly, in line with the lower immune responses induced when using HI-Ro or Frac-Ro (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), pigs vaccinated with these adjuvants showed a notable drop in the vaccine efficacy. Indeed, only 2 out of 6 pigs (33.3%) from each group inoculated with HI-Ro or Frac-Ro survived the challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Moreover, only one of these four surviving pigs showed robust protection, while the other three pigs presented moderate to severe ASF clinical signs and temperatures exceeding 41\u0026ordm;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-d). The decline in the efficacy of adjuvanted vaccinations was also reflected by an uncontrolled viremia. Indeed, only one surviving pig was negative until the end of the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Importantly, all pigs vaccinated with any of the two adjuvants, including the surviving ones, showed high levels of virus shedding in nasal swabs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. This uncontrolled virus replication was further validated analysing virus loads in lungs and gastrohepatic lymph nodes by qPCR, which were positive in all pigs vaccinated with adjuvant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-d). These results were in contrast with the protection afforded in not adjuvanted BA71ΔCD2 vaccinated pigs, which did not show high systemic or local virus loads (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c), demonstrating that HI-Ro or Frac-Ro hamper BA71ΔCD2-induced protective immunity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e4. Reduced BA71ΔCD2 vaccine efficacy mediated by HI-Ro and Frac-Ro is associated with a hampered replication capacity in stimulated macrophages.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe have previously demonstrated that HI-Ro has anti-viral properties, reducing the replication capability of virulent ASFV and PRRSV strains in macrophages\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Thus, the drop in adjuvanted BA71ΔCD2 vaccine efficacy might result from the lower antigen doses generated due to the impaired LAV replication, which is not compensated by the enhanced functionality of BA71ΔCD2-infected macrophages upon HI-Ro or Frac-Ro treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To explore this hypothesis, we measured \u003cem\u003ein vitro\u003c/em\u003e the replication kinetics of BA71ΔCD2 in PAMs treated or untreated with HI-Ro or Frac-Ro. Percentages of infected cells were quantified by time-lapse analysis of macrophages infected with a BA71ΔCD2 expressing a fluorescent fusion protein of the late viral structural protein p54 (BA71ΔCD2-mWasabi). Importantly, the presence of either HI-Ro or Frac-Ro resulted in a significant decrease of the replication capacity of the vaccine strain, regardless of the initial virus dose used (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b; \u003cb\u003eSupplementary Fig.\u0026nbsp;4a-b\u003c/b\u003e). These results were further validated by flow cytometry at 48- and 72- hours post-infection, analysing cells positive for the late expressed viral protein p72. Indeed, both HI-Ro and Frac-Ro significantly decreased the percentage of infected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec; \u003cb\u003eSupplementary Fig.\u0026nbsp;4c\u003c/b\u003e). This effect was probably mediated by the activation of macrophages, since similar results were obtained when infecting the cells together with stimulatory components IFN-γ or LPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec; \u003cb\u003eSupplementary Fig.\u0026nbsp;4c\u003c/b\u003e). Indeed, a short stimulation with HI-Ro or Frac-Ro prior to BA71ΔCD2 infection did not modify the replication capacity of the vaccine virus (\u003cb\u003eSupplementary Fig.\u0026nbsp;5\u003c/b\u003e), thus further suggesting that a full activation of macrophages is required for the anti-viral effect. Importantly, also the mean fluorescent intensity of p72 was decreased in BA71ΔCD2-infected and HI-Ro- or IFN-γ-treated cells, indicating the lower replicative capacity of the vaccine virus at the single cell level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed; \u003cb\u003eSupplementary Fig.\u0026nbsp;4d\u003c/b\u003e). In this line, analysis of percentages of infected cells by the detection of the early viral protein p30 also demonstrated the lower replicative capacity of BA71ΔCD2 in the presence of HI-Ro or Frac-Ro (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), denoting that the antiviral activity affects early steps of the virus replication cycle. Notably, these differences did not result from a cytotoxic effect of the treatments, since the percentages of alive cells were not significantly decreased in the presence of HI-Ro or Frac-Ro (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee; \u003cb\u003eSupplementary Fig.\u0026nbsp;4e\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhile waiting for the development of efficient subunit vaccines against ASF, the use of ASFV-based LAVs with improved biosafety profiles might represent a temporary solution in affected areas\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Here, we have shown that any attempt to increase LAVs biosafety by the use of bacterium-derived adjuvants should avoid the triggering of an antiviral state in macrophages. The use of immunostimulants such as HI-Ro and Frac-Ro might counteract the ASFV-mediated inhibition of macrophage functionality, and thus help in the induction of protective immune responses. However, inherent with this enhanced functional state of macrophages was the activation of an antiviral response, which in turn decreased intracellular LAV replication and thus the amount of antigens required to induce protective immunity. Therefore, the use of adjuvants combined with ASFV-based LAVs must target specific immune components and/or signalling pathways promoting innate immune responses, while allowing enough LAV replication to reach the threshold of viral antigen levels required to trigger a protective adaptive immunity.\u003c/p\u003e\u003cp\u003eThe use of LAVs is the only current vaccine strategy conferring solid protection against ASF, as evidenced by the two LAV ASF vaccines registered in Vietnam\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, their implementation in the field is limited due to biosafety concerns\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Indeed, the balance between virus attenuation and immunogenicity is very subtle, with the consequent potential of reversion to virulence\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. However, despite these concerns, there is an open discussion on the potential benefits derived from a rational use of LAVs in areas affected by virulent ASFV strains\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Thus, the optimization of LAVs to improve their biosafety profile may contribute to controlling ASFV spread while reducing the risk of adverse events. In this scenario, the use of adjuvants represents a potential strategy that deserves investigation. Adjuvants boost vaccine-induced immune responses while reducing the amount of antigen required for effective immunization\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Therefore, the combination of adjuvants with ASF LAVs might lead to a reduction of the vaccine dose with the subsequent drop in potential biosafety issues. However, vaccines based on live viruses have a robust endogenous stimulatory capability, and typically do not need adjuvants to trigger immune responses\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. But this concept should be reconsidered for viruses suppressing innate immune signalling pathways to evade antiviral immunity. Indeed, here we have demonstrated that BA71ΔCD2 combined with HI-Ro or Frac-Ro rescues the functionality of infected macrophages, promoting cytokine production and the upregulation of activation markers. These results demonstrate that there is room for adjuvant-mediated enhancement of ASF LAV immunogenicity. The attenuation of ASFV is usually achieved by mutations or deletions in genes involved in immunosuppressive mechanisms\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, such as the CD2v-encoding gene deleted from the BA71ΔCD2\u003csup\u003e35,36\u003c/sup\u003e. However, these genetic modifications in such a complex virus do not completely revert its capability to compromise macrophage functionality\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Thus, it is likely that similar results obtained with the BA71ΔCD2 would be obtained using other ASFV attenuated strains. Finally, this strategy might be also applied to LAVs against porcine reproductive and respiratory syndrome (PRRS) and classical swine fever (CSF) viruses, which also infect macrophages and suppress innate immunity\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe enhanced functionality of BA71ΔCD2-infected macrophages observed \u003cem\u003ein vitro\u003c/em\u003e upon both HI-Ro and Frac-Ro stimulation did not result in an improvement in vaccine efficacy against a lethal challenge \u003cem\u003ein vivo\u003c/em\u003e, regardless of the adjuvant used. Instead, the protection afforded upon administration of the adjuvanted LAVs was significantly reduced. Vaccine efficacy against any pathogen depends on several factors, but the antigen levels and the type and magnitude of the induced immune responses are particularly critical\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In this line, HI-Ro or Frac-Ro adjuvanted BA71ΔCD2 generated lower levels of ASFV-specific humoral and cellular responses than the non-adjuvanted LAV, but did not alter the quality of virus-specific immune responses. Indeed, the results indicate that HI-Ro and Frac-Ro neither significantly modified virus-specific antibody isotypes, nor the ratio between antibody and cellular responses induced in each pig. Thus, it is likely that the decreased vaccine efficacy was linked to the insufficient induction of virus-specific antibodies and T cells, which did not reach the required thresholds to protect the animals. Mechanistically, it is feasible to hypothesise that the lower immunogenicity of the adjuvanted LAV is associated with a reduced replication rate of the BA71ΔCD2 LAV \u003cem\u003ein vivo\u003c/em\u003e, thus not producing the required antigen levels to induce a protective immunity. This hypothesis is supported by the lower capability of the BA71ΔCD2 to replicate \u003cem\u003ein vitro\u003c/em\u003e in the presence of HI-Ro or Frac-Ro, likely as a consequence of the antiviral state induced in activated macrophages. Indeed, ASFV is sensitive to interferon-induced responses, as shown both \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Moreover, an elegant study in a mouse model showed that short-term IFN-I blockade during immunization with several viral vaccines enhances their efficacy through a transient \u0026ldquo;spike\u0026rdquo; in antigen levels\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Thus, the capability of \u003cem\u003eRothia\u003c/em\u003e-derived components to induce the expression of interferon stimulated genes (ISGs) in BA71ΔCD2-infected macrophages might restrain the virus replication rate with the consequent decrease of antigen production. However, we cannot discard other factors being also responsible for the decreased adjuvanted vaccine efficacy, since the events underlying vaccine-induced immunity \u003cem\u003ein vivo\u003c/em\u003e are highly complex, with many cell subsets involved. This complexity is evidenced by studies performed using adjuvants for live attenuated influenza virus vaccines. In mice, a-C-GalCer increased influenza-specific humoral and cellular responses in a Natural killer T cell-dependent manner, and subsequently reduced both morbidity and mortality after challenge\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, in line with our results, a-C-GalCer impaired the replication of the live vaccine in pigs, reducing vaccine-induced cross-protective immune responses\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Thus, further \u003cem\u003ein vivo\u003c/em\u003e studies not restricted to LAV-macrophage interactions analyses \u003cem\u003ein vitro\u003c/em\u003e are required to understand the adjuvant-triggered mechanisms directly modulating vaccine responses.\u003c/p\u003e\u003cp\u003eThe results discussed above clearly indicate that a fine-tuned and rational selection of adjuvants is necessary to exploit their use for ASF LAVs. Thus, to obtain an adjuvanted vaccine formulation with higher biosafety profiles, further research is needed to better characterize the innate immune responses associated with a favourable outcome upon ASFV or LAV infections. In this context, several early correlates of protection have been recently identified upon immunization with the incompletely attenuated ASFV strain Estonia 2014\u003csup\u003e45\u003c/sup\u003e. In this model, the prompt activation of antigen presenting cells (APC), plasma cells and T cells correlated with the induction of protective immunity. However, a systemic antiviral response and high levels of IFNa in serum were also identified as favourable parameters. These results contrast with our association of an antiviral response in macrophages with a decreased vaccine efficacy. However, this study analysed systemic responses in blood in a model characterised by long viremia, and infection outcomes are usually linked to early events occurring at the site of initial viral replication\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This is especially important for completely attenuated ASFV strains such as BA71ΔCD2 and other LAVs, which usually do not spread systemically or have short viraemic periods\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Thus, further studies are required to identify early and local correlates of protection induced by LAV, which will help to decipher the innate immune components that need to be targeted by adjuvants. Rationally selected adjuvants should achieve a good balance between an enhanced immune functionality in innate immune cells, and the capability of the vaccine virus to replicate and produce the required antigen levels to trigger a protective adaptive immune response.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003ePreparation of heat-inactivated\u003c/b\u003e \u003cb\u003eR. nasimurium\u003c/b\u003e \u003cb\u003e(HI-Ro) and cell-free supernatant (Frac-Ro).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGrowth of \u003cem\u003eR. nasimurium\u003c/em\u003e to obtain the required multiplicity of infection (MOI) was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The bacterium was quantified by serial dilutions and plating, and inactivated at 65\u0026deg;C for 1 hour. Inactivation was confirmed by absence of overnight growth on chocolate agar at 37\u0026deg;C with 5% of CO\u003csub\u003e2\u003c/sub\u003e. To obtain the cell-free supernatant, a plate with a \u003cem\u003eR. nasimurium\u003c/em\u003e overnight culture was washed using 10 mL of phosphate-buffered saline (PBS), and centrifuged at 2.500 x g 10 min. The cell-free resulting supernatant was sterilized using a 0.2 \u0026micro;m filter. The different fractions from the supernatant were obtained using centrifugal filters with a molecular weight cut-off (MWCO) of 100 or 10 kDa (Amicon UFC9100, UFC9010). Supernatant fractions of \u0026ge;\u0026thinsp;100, 100\u0026thinsp;\u0026minus;\u0026thinsp;10, \u0026le;\u0026thinsp;10 MWCO kDa were recovered with 10 mL of Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco). For \u003cem\u003ein vitro\u003c/em\u003e stimulatory assays, HI-Ro was first centrifuged at 2500 x g 10 min and resuspended in the corresponding media at MOI 50. Frac-Ro was diluted 1:2 in the corresponding x2 concentrated media. For \u003cem\u003ein vivo\u003c/em\u003e experiments, HI-Ro was used as 10\u003csup\u003e7\u003c/sup\u003e cfu/mL and Frac-Ro was diluted 1:2 with PBS.\u003c/p\u003e\n\u003ch3\u003eViruses\u003c/h3\u003e\n\u003cp\u003eThe virulent ASFV strain Georgia2007/1 (genotype II) was kindly provided by Dr. Linda Dixon (WOAH reference laboratory, Pirbright Institute, UK), growth in PAMs, and titrated by hemadsorption assay\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The BA71ΔCD2 is a live attenuated strain lacking the CD2v gene (EP402R) obtained by homologous recombination from the parental virulent BA71 strain\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. It was grown in the stabilised COS-1 cell line (ATCC), and titrated by immunoperoxidase monolayer assay (IPMA)\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Titres were calculated by the Reed and Muench method, and expressed as 50% haemagglutination activity units (HAU\u003csub\u003e50\u003c/sub\u003e)/mL or 50% tissue culture infectious dose. TCID\u003csub\u003e50\u003c/sub\u003e titres obtained from IPMA were converted to plaque forming units (pfu) applying the Poisson distribution (pfu/mL\u0026thinsp;\u0026asymp;\u0026thinsp;0.7 \u0026times; TCID\u003csub\u003e50\u003c/sub\u003e/mL).\u003c/p\u003e\n\u003ch3\u003ePorcine alveolar macrophages and cytokine quantification from cell culture supernatants\u003c/h3\u003e\n\u003cp\u003ePorcine alveolar macrophages (PAMs) were obtained through lung lavage of healthy animals (from Landrace, Landrace x Duroc and Landrace x Large White pig breeds) as previously described\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen), 1% of penicillin-streptomycin/mL (P/S) (Invitrogen), 1% of L-glutamine (Invitrogen), and 0.5% of nystatin (Invitrogen). For \u003cem\u003ein vitro\u003c/em\u003e stimulation assays, three to four lots of PAMs were seeded in 96-well flat-bottom plates at 5-6x10\u003csup\u003e5\u003c/sup\u003e cells/well and left overnight at 37\u0026deg;C. Then, cells were incubated with alive \u003cem\u003eR. nasimurium\u003c/em\u003e, HI-Ro, or Frac-Ro at the indicated time points. After stimulation, supernatants were collected and stored at -80\u0026deg;C for TNF (R\u0026amp;D system), IL-1β (R\u0026amp;D system), and IFN-γ (King Fisher) quantification by ELISA, following the manufacturing procedure.\u003c/p\u003e\n\u003ch3\u003eRNA-seq library preparation and sequencing\u003c/h3\u003e\n\u003cp\u003eFour lots of PAMs were seeded at 5x10\u003csup\u003e6\u003c/sup\u003e cells/well in a 6-well flat-bottom plate, and left overnight at 37\u0026deg;C with 5% of CO\u003csub\u003e2\u003c/sub\u003e. PAMs were stimulated for 6 or 24 hours with Frac-Ro (dilution 1/2). Non-stimulated cells were used as control. Total RNA was isolated using the RNeasy Mini Kit (Qiagen) following the manufacturer\u0026rsquo;s protocol. To ensure RNA quality, DNase I treatment was performed for 15 min at room temperature. RNA was sequenced at the Centre Nacional d\u0026rsquo;An\u0026agrave;lisi Gen\u0026ograve;mica (CNAG), Barcelona, Spain. Total RNA concentration was quantified using Qubit RNA BR Assay kit (ThermoFisher Scientific) and the RNA integrity was estimated by Agilent Bioanalyzer. The RNASeq libraries were prepared with KAPA mRNA HyperPrep Kit (Roche) following the manufacturer\u0026rsquo;s recommendations starting with 500 ng of total RNA as the input material. The library was quality controlled on an Agilent 2100 Bioanalyzer with the DNA 7500 assay. The libraries were sequenced on NovaSeq 6000 (Illumina) with a read length of 2x51bp, following the manufacturer\u0026rsquo;s protocol for dual indexing. Image analysis, base calling and quality scoring of the run were processed using the manufacturer\u0026rsquo;s software Real Time Analysis.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eRNA-seq bioinformatic analysis\u003c/h2\u003e\u003cp\u003eIllumina reads were aligned to the \u003cem\u003eSus scrofa\u003c/em\u003e reference genome (Sscrofa11.1) with STAR (v2.7.8a) using ENCODE-recommended parameters\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Gene-level quantification used RSEM (v1.3.0) against Ensembl Sus scrofa annotation release 110\u003csup\u003e52\u003c/sup\u003e. Differential expression was tested with limma (v3.42.3). TMM was applied to compute library-size/compositional scaling factors, and voom converted counts to log2-counts-per-million (logCPM) while modeling the mean\u0026ndash;variance trend on logCPM to derive observation-level precision weights\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Linear models were fit to the weighted logCPM values. Given the paired design, inter-individual effects were modeled with duplicateCorrelation; we extracted pairwise contrasts and the treatment \u0026times; vaccination interaction. Genes were called DE at Benjamini\u0026ndash;Hochberg FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Functional enrichment used DAVID (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://david.ncifcrf.gov/\u003c/span\u003e\u003cspan address=\"http://david.ncifcrf.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e55\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimal experiment\u003c/h3\u003e\n\u003cp\u003eLandrace x Large White breed males were subjected to a seven-day acclimation period prior to vaccination at seven weeks of age. Animals were fed \u003cem\u003ead libitum\u003c/em\u003e. Pigs were divided into four groups of six animals. Three different vaccine formulations were tested: non-adjuvanted vaccine, HI-Ro-adjuvanted vaccine [10\u003csup\u003e7\u003c/sup\u003e cfu/mL], and Frac-Ro-adjuvanted vaccine [dilution 1:2]. In all cases, vaccinated animals were intranasally inoculated with 10\u003csup\u003e4\u003c/sup\u003e pfu/animal of the attenuated LAV prototype BA71ΔCD2. Each animal received 2 mL of the vaccine diluted in PBS via intranasal inoculation (1 mL/nostril). The remaining six animals were used as unvaccinated controls and received 2 mL of PBS alone (1 mL/nostril). At day 22 post-vaccination (p.v.), all animals received 2 mL of an intranasal challenge containing 10\u003csup\u003e5\u003c/sup\u003e HAU\u003csub\u003e50\u003c/sub\u003e of the highly virulent ASFV strain Georgia2007/1 (1 mL/nostril). Rectal temperature and clinical signs were monitored daily during all the experiments as previously described\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Sera and nasal swabs were taken at 0, 7, 15, and 22 days p.v., and 0, 7, 13, and 20 days post-challenge (p.c.) to quantify viral loads by qPCR and ASFV-specific antibodies by ELISA. PBMCs were isolated at day 22 p.v. (day 0 p.c.) to measure ASFV-specific IFN-γ-producing cells by ELISpot. Finally, on the last day of the study lung and gastrohepatic lymph node tissues from each animal were isolated and kept at -80 until analysis.\u003c/p\u003e\n\u003ch3\u003eQuantitative PCR for the detection of ASFV\u003c/h3\u003e\n\u003cp\u003eASFV loads in sera, nasal swabs, and tissues were assessed by SYBR Green qPCR targeting the ASFV serine protein kinase gene (R298L; PK) as previously described\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Briefly, the viral genomic DNA was obtained using IndiMag\u0026reg; Pathogen Kit (INDICAL Bioscience) in a semi-automated manner by using a KingFisher System (Thermo) according to the manufacturer\u0026rsquo;s instructions. qPCR amplifications were performed in duplicates using the corresponding standards for absolute quantification. The results were expressed as log\u003csub\u003e10\u003c/sub\u003e genome-equivalent copies (GEC) per millilitre of sera or nasal swab, or per 0.1 g of tissue. The detection limit of the technique was 10\u003csup\u003e3\u003c/sup\u003e GEC/mL.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e\u003cp\u003eASFV-specific antibodies in pig sera were detected by the WOAH-approved ELISA based on soluble extracts from ASFV-infected cells\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Samples were serially diluted from 1/100 to 1/1.562.500 to calculate the endpoint-titration, with a cut-off defined as the average negative control plus three times the standard deviation of the negative control. Positive sera were detected using the secondary peroxidase-conjugate antibodies: rabbit anti-pig IgG at 1/20000 dilution (Sigma-Aldrich), anti-pig IgG1 at 1/1000 (Bio-Rad), anti-pig IgG2 at 1/1000 (Bio-Rad), or anti-pig IgM at 1/100000 (BioRad). Soluble 3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethylbenzidine (TMB, Sigma-Aldrich) was used as a specific peroxidase substrate. H2SO4 at 1N was used as a stop solution and plates were read at 450nm. All samples were run in technical duplicates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eIFN-γ enzyme-linked immunosorbent spot (ELISpot) assay\u003c/h2\u003e\u003cp\u003eThe IFN-γ-ELISpot assay was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Briefly, IFN-γ-secreting cells in PBMCs were measured by ELISpot assay using the purified mouse anti-pig IFN-g (clone P2G10, BD Pharmingen) as capture antibody and biotinylated mouse anti-porcine IFN-g antibody (clone P2C11, BD Pharmingen) as detection antibody. PBMCs were stimulated with BA71ΔCD2 or Georgia2007/1 at a MOI of 0.2, and incubated for 16 hours at 37˚C, 5% CO2. Sample scoring\u0026thinsp;\u0026ge;\u0026thinsp;300 spots/million PBMCs received a score of 300. Samples approaching this ceiling were not diluted further, so values at the cap may underestimate true spot counts.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eLuminex-based multiplex assay\u003c/h2\u003e\u003cp\u003ePAMs were stimulated for 24 hours with the three cell-free supernatant fractions and supernatants were recovered and kept at -80\u0026ordm;C until analysis. Cytokine levels were quantified using the Luminex xMAP technology following the manufacturer\u0026rsquo;s instructions. Measurements included IFN-α, IL-1b, IL-4, IL-6, IL-12, and IL-10, IL-12p40 (ProcartaPlex Porcine Cytokine \u0026amp; Chemokine Panel 1; Thermo-Fisher Scientific). Concentrations of each cytokine were calculated using the xPONENT software (Luminex).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMicrofluidic quantitative PCR assay\u003c/h2\u003e\u003cp\u003eFive lots of PAMs were seeded at 6x10\u003csup\u003e5\u003c/sup\u003e cells/well in a 48-well flat-bottom plate and left overnight at 37\u0026deg;C with 5% of CO\u003csub\u003e2\u003c/sub\u003e. Cells were infected during 2h with the BA71ΔCD2 ASFV strain at MOI 0.1 in 100 \u0026micro;l/well of RPMI supplemented with 1% L-glutamine. After infection, cells were stimulated overnight with HI-Ro or Frac-Ro. Total RNA was isolated using the RNeasy Mini Kit (Qiagen) following the manufacturer\u0026rsquo;s protocol. Concentration was quantified using Qubit RNA BR Assay kit (ThermoFisher Scientific) and the RNA integrity was estimated by Agilent Bioanalyzer. cDNA was obtained from 150 ng of total RNA using the PrimeScript RT reagent Kit (Takara, Japan) following the manufacturer\u0026rsquo;s instructions. Primer design (\u003cb\u003eSupplementary Data 2\u003c/b\u003e) and validation was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Gene expression levels were measured in duplicates using a microfluidic qPCR with the 48.48 Dynamic Array integrated fluidic circuit of the Biomark HD system (Fluidigm Corporation). Data was analyzed applying the relative standard curve method and using the Fluidigm Real-Time PCR analysis software 4.1.3 and the DAG expression software 1.0.5.6\u003csup\u003e60\u003c/sup\u003e. Target gene expression levels were normalized against the average of three reference control genes (YWHAZ, RPL4 and GAPDH), and z-score normalized values were represented in a Heatmap.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eTime-lapse microscopy of viral infection kinetics\u003c/h2\u003e\u003cp\u003eThe BA71ΔCD2-mWasabi fluorescent virus, encoding the mWasabi fluorescent protein (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fpbase.org/protein/mwasabi/\u003c/span\u003e\u003cspan address=\"https://www.fpbase.org/protein/mwasabi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e fused to the C-terminus of the p54 ASFV protein, was generated by CRISPR technology as previously described\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To evaluate the capability of BA71ΔCD2 to replicate in the presence of HI-Ro and Frac-Ro, PAMs were seeded at 1.5x10\u003csup\u003e5\u003c/sup\u003e cells/well and infected with the BA71ΔCD2-mWasabi at MOI 0.1 or 3.5 in RPMI supplemented with 1% L-glutamine for 2 hours. Next, cells were treated with HI-Ro or Frac-Ro. Viral infection kinetics were then quantified over 72 hours by time-lapse microscopy using an IncuCyte\u0026reg; SX5 (Sartorius BioAnalytical Instruments Inc, CA, USA). Specifically, plates were imaged every 2h at 20x (4 fields of view per well) using the device\u0026rsquo;s \u0026ldquo;AI Scan\u0026rdquo; module in two channels (phase and green) using default acquisition parameters (green acquisition time 300 ms). Cell detection was performed using the \u0026ldquo;AI Cell Health\u0026rdquo; module (Segmentation Sensitivity\u0026thinsp;=\u0026thinsp;0.6) without filtering for cell size. Classification of detected cells across the dimensions \u0026ldquo;uninfected/infected\u0026rdquo; was finally performed using the \u0026ldquo;Cell-by-Cell Classification\u0026rdquo; module: infected cells were identified based on a Green Mean Intensity threshold of 0.6 GCU (set empirically to have ~\u0026thinsp;99% of cells in uninfected controls shown as negative).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry\u003c/h2\u003e\u003cp\u003ePAMs were seeded at 6x10\u003csup\u003e5\u003c/sup\u003e cells/well in a 48-well flat-bottom plate infected with BA71ΔCD2 at MOI 0.1 for 2 hours in 50 \u0026micro;l/well of RPMI supplemented with 1% L-glutamine for. Next, cells were treated with HI-Ro or Frac-Ro. At 48 h post-infection, cells were stained for viability with LIVE/DEAD Fixable Violet Dead Cell Stain Kit following the manufacturer\u0026rsquo;s instructions (ThermoFisher Scientific). Blockage of Fc receptors was performed with PBS containing 5% of porcine serum (Gibco) for 15 min on ice prior to antibody staining. For intracellular staining of virus-infected PAMs, cells were fixed and permeabilized with the BD Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer\u0026rsquo;s protocol, and incubated during 30 min on ice in Perm/Wash buffer with anti-p72 antibody (Eurofins Ingenasa; M.11.PPA.I1BC11) or anti-p30 antibody (hybridoma 1E12F5H2) kindly provided by M. Dom\u0026iacute;nguez for ASFV. Then, cells were incubated with the secondary antibodies anti-mouse IgG1 (eBioscience; 25-4015-82) and anti-mouse IgG2a (Thermo fisher; A-21134), respectively. In all cases, samples were acquired in a BD FACSAria IIu flow cytometer (BD Biosciences) and data was analysed using FlowJo v10.8.1 software (Tree Star Inc).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eGraphics were created and analysed using the Prism version 8.0.2 software (GraphPad), and RStudio. Each statistic test is indicated in the corresponding figure legend. Statistical difference was set up at: ns p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; *p \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\le\\:\\)\u003c/span\u003e\u003c/span\u003e 0.05; **p \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\le\\:\\)\u003c/span\u003e\u003c/span\u003e 0.01; ***p \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\le\\:\\)\u003c/span\u003e\u003c/span\u003e 0.001; ****p \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\le\\:\\)\u003c/span\u003e\u003c/span\u003e 0.0001.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003eAnimal care and procedures were conducted following the guidelines of the Good Experimental Practice and with the acceptance of the Ethics Committee on Animal Experimentation of the \u003cem\u003eGeneralitat de Catalunya\u003c/em\u003e (protocol code: 12121, approved on 2024/10/09). All experiments were performed in the biosafety level 3 facilities at \u003cem\u003eCentre de Recerca en Sanitat Animal\u003c/em\u003e (IRTA-CReSA, Barcelona).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interest:\u003c/h2\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was funded by the Spanish Ministry of Science and Innovation, MICIU/AEI/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.13039/501100011033\u003c/span\u003e\u003cspan address=\"10.13039/501100011033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, grant PID2022-136312OB-I00 (F.R. and J.A.).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor Contributions: Designed the research: F.R. and J.A. Performed the research: A.T-M., S.M-T., D.M-M., J.M-B, Y.Z., M.J.N., M.M., P.M., J.G-O., B.M-M., E.V., A.C. Analysed and interpreted data: J.A., F.R., E.G-F., S.P., F.A., V.A., A.E-C. Wrote the manuscript: J.A., A.T-M. Reviewed the manuscript: J.A., A.E-C., F.R. All authors have approved the submitted version.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Animal Facility and Clinical and preclinical studies units from IRTA-CReSA for their technical support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eSequence data that support the findings of this study have been deposited in the Gene Expression Omnibus repository: https://www.ncbi.nlm.nih.gov/geo/, code GSE288520.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePortugal, R. et al. Six adenoviral vectored African swine fever virus genes protect against fatal disease caused by genotype I challenge. \u003cem\u003eJ. 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DAG expression: high-throughput gene expression analysis of real-time PCR data using standard curves for relative quantification. \u003cem\u003ePLoS One\u003c/em\u003e 8, e80385 (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7815447/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7815447/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhile the development of effective subunit vaccines against African swine fever (ASF) is ongoing, live attenuated vaccines (LAVs) remain the only current strategy capable of inducing robust protective immunity. However, potential biosafety concerns limit their implementation in the field. Thus, further research is required to develop optimized LAVs with better biosafety profiles. Both the ASF virus (ASFV) and derived LAVs suppress innate immune responses of macrophages, thereby limiting their contribution to the induction of protective immune responses. We hypothesized that adjuvants could restore the functionality of LAV-infected macrophages, allowing for a reduction in the vaccine\u0026rsquo;s effective dose and consequently minimizing the risk of adverse events. To test this hypothesis, we intranasally vaccinated pigs with a suboptimal dose of the LAV BA71ΔCD2, either alone or in combination with two adjuvants derived from the immunostimulatory bacterium \u003cem\u003eRothia nasimurium\u003c/em\u003e. The two immunostimulants enhanced the responsiveness of BA71ΔCD2-infected macrophages, which acquired features of antigen presenting cells. However, both adjuvants reduced the levels of ASFV-specific humoral and cellular responses induced by BA71ΔCD2, consequently decreasing the level of protection against a lethal challenge. Further \u003cem\u003ein vitro\u003c/em\u003e analyses demonstrated that adjuvant-activated macrophages acquired an antiviral state, thereby reducing the replication capability of the LAV. Thus, the adjuvant-mediated decline in vaccine efficacy might result from a lower antigen production by infected cells. These results demonstrate that the use of adjuvants combined with ASFV-based LAVs will require a fine-tune manipulation of macrophages, enhancing their functionality while avoiding a significant inhibition of virus replication, reaching the required balance between the levels of viral antigens and innate immune responses to trigger a protective adaptive immunity.\u003c/p\u003e","manuscriptTitle":"Adjuvant-induced macrophage activation compromises BA71ΔCD2-mediated protection against African swine fever virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-06 18:17:36","doi":"10.21203/rs.3.rs-7815447/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-30T06:28:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-18T14:09:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T03:49:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68003703256280214624860098881137309194","date":"2025-12-01T10:01:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327466331607696581152370072654562860741","date":"2025-11-25T09:22:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-24T03:22:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53951347958886751595182366237612177790","date":"2025-11-09T10:39:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-27T20:45:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-27T20:40:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-19T10:51:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2025-10-09T09:02:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"337ac873-da04-4586-ae82-c401a272cedf","owner":[],"postedDate":"November 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56998885,"name":"Biological sciences/Immunology"},{"id":56998886,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-27T16:06:47+00:00","versionOfRecord":{"articleIdentity":"rs-7815447","link":"https://doi.org/10.1038/s41541-026-01461-5","journal":{"identity":"npj-vaccines","isVorOnly":false,"title":"npj Vaccines"},"publishedOn":"2026-04-24 15:59:17","publishedOnDateReadable":"April 24th, 2026"},"versionCreatedAt":"2025-11-06 18:17:36","video":"","vorDoi":"10.1038/s41541-026-01461-5","vorDoiUrl":"https://doi.org/10.1038/s41541-026-01461-5","workflowStages":[]},"version":"v1","identity":"rs-7815447","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7815447","identity":"rs-7815447","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}