TYPE Original Research PUBLISHED 14 June 2023 DOI 10.3389/fimmu.2023.1205080 Enhanced antiviral immunity and OPEN ACCESS dampened inflammation in llama EDITED BY Avijit Dutta, Chang Gung University, Taiwan lymph nodes upon MERS-CoV REVIEWED BY Tesfaye Gelanew, sensing: bridging innate and Armauer Hansen Research Institute (AHRI), Ethiopia Alak Manna, adaptive cellular immune Mayo Clinic Florida, United States *CORRESPONDENCE responses in camelid reservoirs Júlia Vergara-Alert julia.vergara@irta.cat Jordi Rodon 1,2†, Nigeer Te 1,2†, Joaquim Segalés 1,3, † PRESENT ADDRESSES Jordi Rodon, Júlia Vergara-Alert 1,2* and Albert Bensaid 1,2 Institute of Virology, Charité- 1 Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal Universitätsmedizin Berlin, Corporate (CReSA), Catalonia, Spain, 2 IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal Member of Freie Universität Berlin and (CReSA), Catalonia, Spain, 3 Departament de Sanitat i Anatomia Animals, Facultat de Veterinaria, Humboldt-Universität zu Berlin, Berlin, Universitat Autònoma de Barcelona (UAB), Catalonia, Spain Germany Nigeer Te, School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, Hong Kong SAR, China Middle East respiratory syndrome coronavirus (MERS-CoV) infection can cause RECEIVED 13 April 2023 fatal pulmonary inflammatory disease in humans. Contrarily, camelids and bats ACCEPTED 30 May 2023 are the main reservoir hosts, tolerant for MERS-CoV replication without suffering PUBLISHED 14 June 2023 clinical disease. Here, we isolated cervical lymph node (LN) cells from MERS-CoV CITATION Rodon J, Te N, Segalés J, Vergara-Alert J convalescent llamas and pulsed them with two different viral strains (clades B and and Bensaid A (2023) Enhanced antiviral C). Viral replication was not supported in LN, but a cellular immune response was immunity and dampened inflammation mounted. Reminiscent Th1 responses (IFN-g, IL-2, IL-12) were elicited upon in llama lymph nodes upon MERS-CoV sensing: bridging innate and adaptive MERS-CoV sensing, accompanied by a marked and transient peak of antiviral cellular immune responses in responses (type I IFNs, IFN-l3, ISGs, PRRs and TFs). Importantly, expression of camelid reservoirs. Front. Immunol. 14:1205080. inflammatory cytokines (TNF-a, IL-1b, IL-6, IL-8) or inflammasome components doi: 10.3389/fimmu.2023.1205080 (NLRP3, CASP1, PYCARD) was dampened. The role of IFN-l3 to counterbalance COPYRIGHT inflammatory processes and bridge innate and adaptive immune responses in © 2023 Rodon, Te, Segalés, Vergara-Alert camelid species is discussed. Our findings shed light into key mechanisms on and Bensaid. This is an open-access article distributed under the terms of the Creative how reservoir species control MERS-CoV in the absence of clinical disease. Commons Attribution License (CC BY). The use, distribution or reproduction in other KEYWORDS forums is permitted, provided the original author(s) and the copyright owner(s) are camelids, cytokines, immune responses, llama, lymph node, lymphocyte, Middle East credited and that the original publication in respiratory syndrome coronavirus (MERS-CoV) this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Introduction The Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pneumonia and is associated with a high case-fatality rate in humans (1). Currently, clade B strains have a high incidence in the Arabian Peninsula, while clade C strains, restricted to Africa, are not causing outbreaks despite that reactive virus-specific T-cells were found in humans (2). MERS-CoV was reported to abortively infect human T cells in vitro and concomitantly induce apoptotic pathways (3), which might explain the severe lymphopenia Frontiers in Immunology 01 frontiersin.org Rodon et al. 10.3389/fimmu.2023.1205080 commonly reported in MERS patients (4, 5). Altogether, these samples used in this work were obtained during necropsy findings could lead to aberrant or delayed induction of antiviral T procedures of previous studies (31, 32), approved by the Ethical cell responses, as observed in acute phase patients (6–9), and and Animal Welfare Committee of IRTA (CEEA-IRTA) and by the contribute to the high pathogenicity of MERS-CoV. Even so, Ethical Commission of Animal Experimentation of the recovered patients mount effective T cell responses that play a Autonomous Government of Catalonia (file No. CEA-OH/ major role in the outcome of MERS. Remarkably, virus-specific 10942/1). CD8+ T cell responses were also developed by all survivors studied, including those with undetectable antibody responses (10), suggesting that convalescent patients would trigger early Cell culture and MERS-CoV protective immune responses upon a subsequent MERS-CoV infection. Moreover, the crucial role of T cell responses to Vero E6 cells (CRL-1586, ATCC, USA) were cultured in counteract MERS-CoV infection was quickly unravelled in animal Dulbecco’s modified Eagle medium (DMEM; Lonza, Switzerland) model studies. Contrary to B-cell deficient and control animals, supplemented with 5% FCS, 100 U/mL penicillin, 100 µg/mL viral persistence was reported in the lungs of T-cell deficient mice streptomycin, and 2 mM glutamine. (11). Thus, development of robust and functional T cell responses is A passage-3 MERS-CoV Qatar15/2015 strain (clade B; required to fully achieve MERS-CoV clearance. GenBank accession no. MK280984.2) stock and a passage 6 of the MERS-CoV is transmitted to humans by dromedary camels, MERS-CoV Egypt/2013 strain (clade C; GenBank accession no. the main reservoir host (12–14), although other camelid species KJ477103) were prepared as previously described (31, 32). are also susceptible to viral infection (15–19). These species only Infectious virus titers were assayed in Vero E6 cells and develop a subclinical infection, which typically show upper determined by the dilution causing 50% of cytopathic effect in respiratory tract replication and abundant MERS-CoV cell cultures (50% tissue culture infectious dose endpoint, TCID50). shedding (14, 20, 21). Camelids elicit strong innate immune responses with dampened inflammation at the mucosal level (22, 23) similar to those described in bat cells (10, 24, 25). Indeed, Animal infection and sampling bats are tolerant to many viruses including MERS-CoV-like viruses (26) and can be experimentally infected with MERS- Two animals were experimentally infected with the Qatar15/ CoV without suffering from disease (27). These two reservoir 2015 strain (31) and two other llamas with the Egypt/2013 strain species can be reinfected (26, 28), allowing viral maintenance (32). Infection was monitored for 3 weeks. Nasal swabs samples and eventual spread. Therefore, adaptive immune responses were obtained daily until 15 days post inoculation (dpi), plus at 17 must be important determinants of host disease resistance but and 22 dpi. Sera samples were obtained before MERS-CoV do not interrupt viral maintenance within these animal challenge and at 7, 14 and 22 dpi, when animals were euthanized populations. Indeed, protective humoral immune responses and necropsied. against MERS-CoV are known to occur in camelids after natural and experimental infection (15–20, 29). Efficient antigen presentation in draining lymph nodes (LN) is essential Cervical lymph node cell isolation to ensure successful induction of specific T and B cell adaptive immune responses. Previous experimental studies have shown At necropsy day, cervical LNs were collected in Roswell Park the presence of infectious MERS-CoV in LN of dromedary Memorial Institute 1640 medium (RPMI, Lonza, Switzerland) camels (20, 21). Moreover, in llamas, abundant nucleoprotein supplemented with 100 U/mL penicillin, 100 µg/mL antigen was observed within dendritic-like cells in cervical LNs streptomycin, 2 mM glutamine and 10% FCS (all ThermoFisher at 4 days post experimental inoculation (dpi) and MERS-CoV Scientific, USA) and kept at 4°C until transferred to the lab. LNs RNA persisted until 24 dpi (30). Although no tissue damage was were mechanically disaggregated with a sterile blade. Cells were observed, it is unclear whether the virus could replicate in these filtered through 70 µm strainers (Corning, USA) and concentrated lymphoid organs. In the present study, we mimicked a secondary by centrifugation. Red blood cells were removed using ACK lysing exposure to MERS-CoV clade B and C strains in vitro in cervical buffer (ThermoFisher Scientific, USA), according to the LN cells from previously inoculated llamas to investigate viral manufacturer’s instructions. LN cells were resuspended and replication and cellular immune responses at the transcriptional level. cultured in RPMI supplemented with 10% fetal calf serum (FCS; EuroClone, Italy), 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM glutamine and 5×10 -5 M b-mercaptoethanol (Sigma- Methods Aldrich, USA). Animal welfare and ethics MERS-CoV exposure to lymph node cells Animal and laboratory experiments with MERS-CoV were performed at the biosafety level-3 (BSL-3) facilities of the After isolation, LN cells were cultured in triplicates. One million Biocontainment Unit of IRTA-CReSA (Barcelona, Spain). Animal cells were seeded onto 24-well plates in 1 mL final volume of cell Frontiers in Immunology 02 frontiersin.org Rodon et al. 10.3389/fimmu.2023.1205080 culture medium alone (mock) or containing MERS-CoV Qatar15/ Relative quantification and data analysis 2015 or Egypt/2013 strain (MOI of 0.1) and cultured for 48 h at 37° C and 5% CO2. Culture supernatants and cells were collected at 0, Gene expression analyses were performed as previously 24 and 48 hours post-exposure (hpe). Additional fresh control LN described (35). Briefly, data were collected with the Fluidigm cells were also collected prior culture. Real-Time PCR Analysis software 4.1.3 (Fluidigm Corporation, USA) and analyzed with the DAG expression software 1.0.5.6 (37). The relative standard curve method (see Applied Biosystems Viral and cellular RNA extraction user bulletin #2) was applied to compare gene expression levels of LN cells cultured in different conditions against those of freshly Viral RNA was extracted from supernatant samples using the prepared LN cells, using multiple reference gene normalization IndiMag pathogen kit (Indical Biosciences, Germany) and a (GAPDH, HPRT1 and UbC). Relative expression of IFN-l1 and Biosprint 96 workstation (Qiagen, Germany), according to the IFN-l3 was calculated according to the 2-DDCT method (38), using manufacturer’s instructions. Total RNA was extracted from llama the same normalizer genes, since expression levels of these genes in LN cells using the Direct-zol RNA Miniprep (Zymo research, USA), control samples were too low to generate standard curves. Relative following the manufacturer’s protocol. After RNA extraction, an expression of each gene in a particular sample was expressed in mean additional HL-dsDNase treatment using the Heat&Run gDNA fold-change values (Fc) and are shown in Supplementary Table 1. removal kit (ArcticZymes Technologies, Norway) was performed The unpaired t-test was used to statistically compare the relative according to the manufacturer’s protocol to completely remove the expression levels of genes from LN cells exposed to MERS-CoV reminiscent genomic DNA. Finally, 1 U/µL RNase inhibitors Qatar15/2015, Egypt/2013 and those of cells cultured in media only. (Invitrogen, Life Technologies, Waltham, USA) were added. All statistical analyses were performed using GraphPad Prism 9.3.1 Samples were stored at -75°C until further analyses. The purity (GraphPad Software, USA). Differences were considered significant and quantity of the extracted RNA were assessed using a BioDrop at p-values < 0.05. µLITE Spectrophotometer (BioDrop Ltd, UK). A260:A280 ratio ranged from 1.6 to 1.8. Results MERS-CoV RNA detection by RT-qPCR Four llamas were primed by experimental inoculation with MERS-CoV Qatar15/2015 (n = 2) or Egypt/2013 (n = 2) strains, Viral genomic RNA was detected in culture supernatant by causing productive infection resolved at 8 to 9 dpi. None of the performing the UpE RT-qPCR assay (33), with minor modifications inoculated llamas displayed clinical signs along the study. Genomic as previously described (15, 29, 34). Samples with a quantification and subgenomic viral RNA were detected for both strains at similar cycle (Cq) value ≤40 were considered positive. levels in nasal swabs (Supplementary Figure 1A, B). Thus, llamas shed high titers of infectious virus independently of the strain causing infection (Supplementary Figure 1C). Animals from both cDNA synthesis groups seroconverted to MERS-CoV with similar levels of nAbs that were detected from 2 weeks after infection onwards One hundred and ten ng of total RNA were converted to cDNA (Supplementary Figure 1D). Overall, llamas followed similar in a final volume of 10 mL using the PrimeScript RT reagent Kit trends in viral shedding and development of humoral responses (Takara, Japan), by a combination of oligo-dT and random regardless of the MERS-CoV strain inoculated. hexamers, following the manufacturer’s instructions. cDNA Three weeks after infection, llama cervical LN were collected samples were stored at -75°C until subsequent use. and their cells cultured in the presence of MERS-CoV for 0, 24 and 48 h, as schematically represented in Figure 1A. Cells were exposed to the same MERS-CoV strain used for priming. We monitored Fluidigm biomark microfluidic RT-qPCR viral titres in culture supernatants and seeded cells. Importantly, MERS-CoV was not found in cervical LN cells at 22 dpi, as Transcription of cytokines and immune-related genes were evidenced by the absence of viral RNA in mock-treated cells quantified using a previously validated protocol to study camelid (Figure 1B, C). Independently of the strain used to pulse cells, immune responses (22, 35). A Fluidigm Biomark microfluidic RT- viral loads in supernatant samples decreased over time, as qPCR assay was used to quantify immune-gene expression of LN determined by RT-qPCR for genomic RNA detection (Figure 1B). cell samples. In addition, specific primers for the quantification of Also, microfluidic RT-qPCR results indicated that cell-associated MERS-CoV subgenomic RNA (M mRNA) were added to the assay MERS-CoV RNA declined (Figure 1C). Therefore, cervical LN cells (36). Amplification reactions were coupled with Tm analyses to of llama did not support MERS-CoV replication at least in vitro. ensure that specific amplifications occurred. Non-template controls We also studied whether LN cells could mount immune were also included in the assays. responses to a secondary viral exposure in vitro. Transcriptomic Frontiers in Immunology 03 frontiersin.org Rodon et al. 10.3389/fimmu.2023.1205080 A B C FIGURE 1 Susceptibility of llama lymph node (LN) cells to MERS-CoV infection. Solid lines indicate mean values and light represent standard deviation intervals. At 22 days after intranasal inoculation of MERS-CoV of llamas, primed LN cells were isolated and pulsed with the same MERS-CoV strain used for animal inoculation, as represented in panel (A). Panels (B) and (C) display data from llama LN cells seeded in triplicates and exposed for 24 and 48 h to MERS-CoV Qatar15/2015 (purple rectangles), Egypt/2013 (orange circles) or cultured in media only (green triangles). Mean values (± SD) of genomic viral RNA detection in culture supernatants (B) and cell-associated viral RNA (C) were monitored throughout the study. Grey dashed lines depict the detection limits of the assays. Cq, quantification cycle; MERS-CoV, Middle East respiratory syndrome coronavirus. profiles from 43 immune response genes were obtained using a inflammatory responses were not induced along the study. These previously described microfluidic RT-qPCR assay (35), which results evidenced that early and transient antiviral cellular immune included the quantification of type I, II and III interferons (IFNs), responses were effectively triggered in LNs of llamas re-exposed to pattern recognition receptors (PRRs), transcription factors (TFs), MERS-CoV. Responses induced by the MERS-CoV Qatar/2015 IFN-stimulated genes (ISGs), cytokines and chemokines involved in strain were significantly more pronounced than those provoked inflammatory responses, among other immune-related genes by the Egypt/2013 strain. (Figure 2A). Afterwards, gene expression levels of LN cells exposed to MERS-CoV and mock-treated samples were compared to those from freshly isolated cells. Mock-exposed cells experienced Discussion a mild increase of immune response genes transcription and this was more evident at 48 h post in vitro culture (Figure 2A, B). IFN-g The present study suggests that cervical LN cells from llamas do expression was significantly up-regulated in MERS-CoV-treated not support MERS-CoV replication in vitro. In agreement, we cells and progressively increased over time (Figure 2A, B). previously described low levels of dipeptidyl-peptidase 4 (DPP4) Although not statistically significant, a stronger induction of IFN- receptor in lymphoid cells of camelids compared to other species g occurred in cells exposed to the Qatar15/2015 strain than the ones (30, 39). Therefore, we employed an MOI of 0.1 to maximize the exposed to the Egypt/2013 (Figure 2B). Expression levels of IL-2 probabilities of LN cells to interact with MERS-CoV. The absence of and IL-12 similarly increased at 24 hpe and subsequently returned an increase in genomic and subgenomic viral RNA over time to basal levels (Figure 2B). On the other hand, an increase of IL-4 supports that MERS-CoV replication did not occur in cultured expression was observed in cells exposed to the MERS-CoV cells. Furthermore, the expression of DPP4 and MERS-CoV Qatar15/2015 but not to Egypt/2013 strain (Figure 2A, B). nucleoprotein did not co-localize in llama cervical LNs in vivo Remarkably, induction of IL-10 mRNA was not detected in any (30). There is no data on the replication of MERS-CoV in LNs of llama cells. Overall, results are reminiscent of a Th1 response other susceptible species. Nonetheless, our findings support the elicited in LN cells after re-exposure to MERS-CoV, regardless of concept that camelid dendritic-like cells carry MERS-CoV to LNs the strain used for stimulation. without active viral replication, and they might be the drivers of Innate immune gene responses were also monitored. potent immune responses that prevent virus spread. Transcription of IFN-l3 was markedly upregulated in cells We investigated if llamas could mount cellular adaptive treated with both MERS-CoV strains, being significantly higher in immune responses to counteract MERS-CoV infection. Indeed, those stimulated with the Qatar15/2015 strain (Figure 2B). LN cells of previously infected llamas and subsequently exposed However, type I IFNs (IFN-a and IFN-b) were only upregulated to the virus in vitro developed an early induction of IL-12 in all in LN cells exposed to the Qatar15/2015 strain at 24 hpe MERS-CoV pulsed cells, suggesting that both Qatar15/2015 and (Figure 2B). Expression of TFs (STAT1 and IRF7), ISGs (CXCL10, Egypt/2013 strains were effectively mounting an immune response MX1, OAS1 and ISG15), and PRRs (RIG-1, MDA-5 and TLR-7) accompanied by an increase of IFN-g over time. The concomitant were enhanced in cells according to levels of IFNs (Figure 2A, B). induction of IL-2 suggested activation of Th1 lymphocytes, similar Thus, the Egypt/2013 strain moderately induced the above- to previous findings in PBMCs from convalescent human patients mentioned genes at 24 hpe, while higher upregulations occurred pulsed with MERS-CoV peptide pools (6, 10). Alternatively, or in in cells exposed to the Qatar15/2015 strain that waned over time for addition, NK cells residing in camelid LN could be responsible for both strains (Figure 2B). In addition, a significant mild upregulation the up-regulation of IFN-g, as previously described in cattle and of TRIM25, CCL3, and IL-15 was mostly observed at 24 hpe by cells human (40, 41). On the other hand, absence of IL-10 up-regulation exposed to the Qatar15/2015 strain (Figure 2A). Importantly, pro- would indicate that Th2 cells were not induced or recalled. Further Frontiers in Immunology 04 frontiersin.org Rodon et al. 10.3389/fimmu.2023.1205080 A B FIGURE 2 Expression of immune response genes by llama lymph node (LN) cells pulsed with MERS-CoV. A microfluidic RT-qPCR assay was used to quantify transcripts of immune-related genes at different h post MERS-CoV exposure (hpe). (A) After relative normalization, mean expression values (triplicates) of llama LN cells exposed for 24 and 48 h to MERS-CoV Qatar15/2015 (purple rectangles), Egypt/2013 (orange rectangles), or cultured in media only (NS, green rectangles) were calculated respective to non-cultured control cells. Mean log2 fold-change expression values of each studied gene are represented in a heat-map plot with colour variations; blue for up-regulated and black for down-regulated gene expression, respectively. Panel (B) display the relative expression values of some differentially regulated genes at 24 and 48 h after exposure to MERS-CoV Qatar15/2015 (purple), Egypt/ 2013 (orange) or cultured in media only (green). Boxes indicate mean expression values and error bars represent SD intervals. Individual relative expression measurements are shown as empty circles. Grey dashed lines display basal expression levels from freshly isolated control cells. *, p-value < 0.05; **, p-value < 0.001; ***, p-value < 0.0001; ****, p-value < 0.00001; ADs, adaptors; IFNs, interferons; ISGs, IFN stimulated genes; LL, llama; NK, natural killer T cells, PRRs, pattern-recognition receptors; TFs, transcription factors; Th1, T-helper 1; Th2, T-helper 2. detailed studies are needed to deeply characterize T- and B- cell dependent inflammatory genes are inhibited under the action of C- responses in LNs. Rel (24). Similar studies should be performed in camelid species to Importantly, significant induction of type I, II and III IFNs was precisely determine mechanisms controlling inflammation and noticed in LN cells of animals primed and re-stimulated in vitro their similarity to those engaged in bats. Nonetheless, as bats, with at least the Qatar15/2015 isolate, with a consequent up- camelids can control inflammation mediating an impaired regulation of ISGs, PRRs and TFs involved in antiviral responses. NLRP3 inflammasome. In the present study, IFN-l3 but not IFN- Strikingly, pro-inflammatory cytokines (TNF-a, IL-1b, IL-6 and IL- l1 was highly up-regulated and might contribute to counterbalance 8), CARD9 (an activator of NF-kB) and components of the the inflammatory effects of type I IFNs (42). Moreover, control of inflammasome (NLRP3, CASP1, PYCARD) remained at basal inflammation is not specific to LN cells, since we previously transcription levels or were slightly up- or down-regulated. described dampened inflammatory responses in the nose, trachea, Differences in these pro-inflammatory cytokines would be and lungs of MERS-CoV-challenged alpacas. Early and transient expected between species, as humans can suffer from severe type I and III IFNs were also produced by the nasal epithelium of MERS but camelids are asymptomatic hosts. This would imply these animals (22, 23). A previous study unravelled the high specific mechanisms of camelids for dampening inflammation as production of type I and III IFNs by human plasmacytoid DCs observed in bats (24, 25). In these virus-tolerant animals, NF-kB- (pDCs) in the absence of productive MERS-CoV replication (43). Frontiers in Immunology 05 frontiersin.org Rodon et al. 10.3389/fimmu.2023.1205080 We hypothesize that camelid pDCs sensing by MERS-CoV could be Author contributions the source of the pronounced IFN-l3 response in LNs. Altogether, our results highlight that IFN-l3 might have a key role in bridging JR, JS, JV-A and AB conceived and designed the study. JR, NT, innate and adaptive immunity from the infected respiratory mucosa JS, JV-A and AB performed the experiments and analyzed the data. to secondary lymphoid organs, as previously described for other All the authors discussed the results. The manuscript was written by viral infections (44, 45). Thus, camelid species own key mechanisms JR and AB. All the authors revised the manuscript. All authors to host MERS-CoV in the absence of clinical disease. contributed to the article and approved the submitted version. At 24 hpe, the Qatar15/2015 strain induced higher antiviral transcripts than the Egypt/2013 strain, while levels of cytokine mRNAs decayed thereafter except for IFN-g. Possibly, pathogen- Funding associated molecular patterns (PAMPs) of the Qatar15/2015 strain better activated type I and III IFN pathways. Alternatively, the peak This study was performed as part of the Zoonotic Anticipation and Preparedness Initiative (ZAPI) [Innovative Medicines initiative of antiviral responses could be elicited earlier with the Egypt/2013 strain. However, our observations should be confirmed with (IMI) grant 115760] and the Veterinary Biocontained research facility Network (VetBioNet) (EU Grant Agreement INFRA- samples from a larger number of animals, being also collected at early time points after viral exposure. Overall, llama cervical LN 2016-1 N°731014) projects, with assistance and financial support from IMI and the European Commission and contributions from cells elicited early antiviral responses in the absence of inflammation to MERS-CoV re-exposure, which were higher for EFPIA partners. JR was partially supported by the VetBioNet. IRTA is supported by CERCA Programme/Generalitat de Catalunya. the clade B strain compared to its clade C counterpart. A potential limitation of our study is the lack of comparison with LN cells from healthy, non-convalescent animals, which may Acknowledgments help to discern unique features of camelid memory T-cell responses versus those occurring in a primary infection. Finally, the use of We thank Dr. Fernando Rodrı́guez from IRTA-CReSA for peptide pools to stimulate camelid LN lymphocytes would reveal critically reviewing the manuscript. We are particularly indebted the most immunogenic MERS-CoV-specific T cell epitopes, and with the staff of the BSL-3 biocontainment facility at CReSA. thus, improve animal vaccine design. In conclusion, the present study suggests that camelid LN cells Conflict of interest could not support MERS-CoV replication. Remarkably, convalescent llamas developed strong cellular antiviral responses The authors declare that the research was conducted in the that are rapidly activated in vitro following a secondary viral absence of any commercial or financial relationships that could be exposure, in the absence of inflammation. construed as a potential conflict of interest. Data availability statement Publisher’s note The original contributions presented in the study are included All claims expressed in this article are solely those of the authors in the article/Supplementary Material. Further inquiries can be and do not necessarily represent those of their affiliated organizations, directed to the corresponding author. or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Ethics statement Supplementary material The animal study was reviewed and approved by Ethical and Animal Welfare Committee of IRTA (CEEA-IRTA) and by the Ethical The Supplementary Material for this article can be found online at: Commission of Animal Experimentation of the Autonomous https://www.frontiersin.org/articles/10.3389/fimmu.2023.1205080/ Government of Catalonia (file No. CEA-OH/10942/1). full#supplementary-material References 1. World Health Organization (WHO). MERS situation update - march 2022 (2022). 3. Chu H, Zhou J, Wong BHY, Li C, Chan JFW, Cheng ZS, et al. 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