Rodon et al. One Health Outlook (2022) 4:12 https://doi.org/10.1186/s42522-022-00068-9 One Health Outlook SHORT REPORT Open Access Protective efficacy of an RBD‑based Middle East respiratory syndrome coronavirus (MERS‑CoV) particle vaccine in llamas Jordi Rodon1,2, Anna Z. Mykytyn3, Guillermo Cantero1,2, Irina C. Albulescu4, Berend‑Jan Bosch4, Alexander Brix5, Jean‑Christophe Audonnet6, Albert Bensaid1,2, Júlia Vergara‑Alert1,2, Bart L. Haagmans3* and Joaquim Segalés1,7*    Abstract Ongoing outbreaks of Middle East respiratory syndrome coronavirus (MERS-CoV) continue posing a global health threat. Vaccination of livestock reservoir species is a recommended strategy to prevent spread of MERS-CoV among animals and potential spillover to humans. Using a direct-contact llama challenge model that mimics naturally occur‑ ring viral transmission, we tested the efficacy of a multimeric receptor binding domain (RBD) particle-display based vaccine candidate. While MERS-CoV was transmitted to naïve animals exposed to virus-inoculated llamas, immuniza‑ tion induced robust virus-neutralizing antibody responses and prevented transmission in 1/3 vaccinated, in-contact animals. Our exploratory study supports further improvement of the RBD-based vaccine to prevent zoonotic spillover of MERS-CoV. Keywords: Animal model, Llama, Camelid, Middle East respiratory syndrome coronavirus, MERS-CoV, Multimeric protein scaffold particles (MPSP), Receptor binding domain (RBD)-based vaccine, Virus transmission, Neutralizing antibodies Main text Dromedaries are the main reservoir, although other MERS-CoV is associated with severe pneumonia and camelid species such as llamas and alpacas are also sus- lethal disease in humans with high case-fatality rates in ceptible to MERS-CoV [3–10]. Camelids, as opposed to the Middle East [1]. The virus still poses a public health humans, undergo a mild to subclinical infection upon concern since ongoing zoonotic transmission events MERS-CoV infection, characterized by upper respiratory from dromedary camels, the main source of infection, tract replication and rapid clearance of the virus within and several major travel-associated outbreaks have been 1–2 weeks after infection [11, 12]. Robust and timely documented [2]. innate immune responses occurring in camelids might play a crucial role in controlling MERS-CoV infection and disease development [4]. Importantly, animals show- ing nasal discharges and asymptomatic carriers shed *Correspondence: b.haagmans@erasmusmc.nl; joaquim.segales@irta.cat abundant quantities of MERS-CoV [3, 5, 11, 12], which 3 Department of Viroscience, Erasmus Medical Centre, Rotterdam, CA 3000, may result in a potential spillover to humans. The Netherlands To date, commercial vaccines and therapeutics against 7 Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, MERS-CoV are lacking, and the World Health Organiza- UAB, Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, Barcelona, Catalonia 08193, Spain tion has advised animal vaccination as a strategy to con- Full list of author information is available at the end of the article trol the spread of MERS-CoV to animals and humans © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Rodon et al. One Health Outlook (2022) 4:12 Page 2 of 5 [13]. Different vaccine prototypes have been tested in in protective efficacy between host species might be camelids to counteract MERS-CoV, all of them focusing explained by the differential response to the vaccine, as on the full-length or specific regions of the spike (S) pro- evidenced by the levels of nAbs elicited [14]. Further, to tein [5, 12, 14, 15]. This protein mediates viral entry by mimic the natural transmission occurring in the field, we binding to the host cell receptor dipeptidyl peptidase-4 previously developed a direct-contact llama transmis- [16] and subsequent fusion of the viral and cellular mem- sion challenge model to demonstrate that a recombinant brane. The spike protein is highly immunogenic and the S1-protein vaccine was able to block MERS-CoV trans- main target of neutralizing antibodies and, therefore, mission among camelids [5]. the antigen of choice for vaccine development against Here, we used the same direct-contact model to MERS-CoV and other betacoronaviruses [17]. Viral-vec- assess the efficacy of a virus-like particle vaccine to tor vaccines expressing the full-length S protein induced block MERS-CoV transmission in llamas. The vac- partial immunity and, in some instances, when exposed cine was composed of self-assembling multimeric to MERS-CoV, reduced rhinorrhea and viral shedding in protein scaffold particles (MPSP) expressing the dromedaries [12, 15]. Importantly, an increase in neu- receptor-binding domain (RBD) of the MERS-CoV S tralizing antibody (nAb) titers was observed after one protein [18]. The MPSP vaccine prototype allows the vaccination of seropositive animals, resulting in mini- self-assembly of antigens into 60-mer particles and mum excretion of viral RNA after exposure to naturally offers enhanced immune responses in comparison to infected camels [15]. This fact is of special relevance due other multivalent and monomeric recombinant vac- to the high prevalence of seropositive camels found in cines [18–20]. Indeed, the proposed vaccine proto- the Middle East. The usage of recombinant protein vac- type induced strong protective immune responses cine candidates based on the S1 subunit have also been that reduced MERS-CoV replication in the upper and proposed for camelids [14]. Three administrations of an lower respiratory tract of experimentally infected rab- S1-based vaccine prototype conferred full protection bits [18]. Since rabbits do not develop severe disease against MERS-CoV in alpacas, as well as delayed and upon MERS-CoV inoculation as occurs in humans, nor reduced infectious viral shedding for 3 days after intra- a subclinical infection with high viral secretions that nasal challenge of dromedary camels [14]. Differences camelid reservoirs experience [21], this study provided Fig. 1 Schematic representation of the experimental design. Two llamas (black) were intranasally inoculated with MERS-CoV (Qatar15/2015) and two days later brought in contact with two naïve (grey) and three vaccinated (red) llamas. Immunization dates are shown in red timeline points and with grey syringes. MERS-CoV-inoculation procedure is stressed as a gold time point. Blood collection days are represented with a red syringe symbol on the weeks scale. Sampling scheme of nasal swabs in all animals is shown using black lines in a daily scale. Dpi, days post-inoculation; i.n., intranasal Rodon et al. One Health Outlook (2022) 4:12 Page 3 of 5 a rationale for testing the MPSP-RBD vaccine proto- shown to replicate efficiently and be transmitted between type in camelids. camelids in direct contact [4, 5]. Clinical signs and body Following a previous experimental design to test vac- temperatures were monitored, and collection of nasal cine efficacy mimicking field-like conditions [5], a group swabs for virological studies were conducted as indi- of three llamas was vaccinated with two doses of the cated in Fig. 1 and detailed in Additional Material and MPSP-RBD in combination with a registered adjuvant Methods. (Fig. 1, Additional Material and Methods). After prime Rectal temperatures of all animals remained basal (37– and boost immunizations, vaccinated (n = 3) and adju- 40 °C) throughout the study (Additional Fig. 2a). None vant-control administrated animals (n = 2) were put in of the inoculated llamas showed clinical signs at any day direct contact with naïve llamas (n = 2) infected with post inoculation (dpi). One contact-control animal showed MERS-CoV (see Additional Fig. 1). Two days before mix- moderate rhinorrhea at 5–9 dpi, and one vaccinated ani- ing the groups together, naïve llamas were inoculated mal from 8 to 19 dpi (Additional Fig. 2b and c, respec- with MERS-CoV Qatar15/2015 strain, a clade B strain tively). As previously reported [5], MERS-CoV-inoculated Fig. 2 MERS-CoV RNA and infectious virus shedding and development of neutralizing antibodies in llamas. Experimentally infected llamas (black) were placed in contact with naïve (grey) and vaccinated (red) animals two days after MERS-CoV inoculation. Genomic (a) and subgenomic (b) viral RNA was quantified in nasal swab specimens collected at different times after MERS-CoV inoculation. Plot (c) show infectious MERS-CoV titres in nasal swabs collected on different days after MERS-CoV inoculation. Plot (d) displays serum neutralizing antibodies elicited against MERS-CoV in vaccinated, experimentally inoculated and in-contact naïve llamas. Each line represents an individual animal. Dashed lines depict the detection limits of the assays. Red and yellow arrows indicate the two MPSP-RBD immunizations and MERS-CoV inoculation days, respectively. Cq, quantification cycle; MERS-CoV, Middle East respiratory syndrome coronavirus; PRNT50, 50% plaque reduction neutralization titre; ­TCID50, 50% tissue culture infective dose Rodon et al. One Health Outlook (2022) 4:12 Page 4 of 5 llamas had detectable genomic and subgenomic viral RNA to prevent animal-to-animal transmission of MERS-CoV in nasal swabs for a period of 2 weeks (Fig. 2a and b) and and, eventually, human spillover, seem limited. shed high titers of infectious virus during the first week after inoculation (Fig. 2c). These animals seroconverted for Abbreviations MERS-CoV and nAbs were detected from 2 weeks after dpi: Days post inoculation; MERS-CoV: Middle East respiratory syndrome infection onwards (Fig. 2d). As determined by RT-qPCR coronavirus; MPSP: Multimeric protein scaffold particles; nAbs: Neutralizing and virus titration in cell culture, MERS-CoV was trans- antibodies; RBD: Receptor-binding domain; S: Spike. mitted to all adjuvant-administered and two out of three vaccinated, in-contact animals at 5–7 dpi (Fig. 2a, b and Supplementary Information c). With the exception of one vaccinated llama, all ani- The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s42522-​022-​00068-9. mals had similar profiles in the duration and levels of viral RNA and infectious virus shedding (Fig. 2a, b and c). These Additional file 1: Fig. 1. Animal distribution scheme inside the experi‑ results are comparable to previous ones obtained in inocu- mental box. Experimental groups were kept in different compartments lated and naïve contact animals [5]; therefore, individual separated by tarpaulin to prevent animal contact. Two days after inocula‑ tion procedure, the tarpaulin was removed and experimentally infected differences observed in the current study may account for llamas (black) were then in direct contact with naïve (grey) and vaccinated minor variations in viral shedding patterns of vaccinated (red) animals. and control-contact animals. The remaining vaccinated- Additional file 2: Fig. 2. Temperature and rhinorrhoea after MERS-CoV contact llama was protected against MERS-CoV infec- exposure to llamas. MERS-CoV experimentally inoculated llamas (black) were, two days later, put in contact with naïve (grey) and vaccinated (red). tion. Only minor traces of MERS-CoV genomic RNA were (a) Rectal temperature was measured daily after MERS-CoV. Each line/ detected in nasal swabs of this animal along the experi- sign represents an individual animal. One naïve (b) and one vaccinated, ment, evidencing its exposure to the virus (Fig. 2a). More- contact animal (c) showed moderate mucus excretion at 5-9 and 8-19 days post-inoculation procedure, respectively. over, subgenomic RNA was not detected at any time point Additional file 3. Materials and methods. of the study in this vaccinated llama and the animal did not shed infectious virus (Fig. 2b and c). Furthermore, all inoc- ulated and in-contact naïve llamas developed a compara- Acknowledgements The authors want to thank Núria Roca from IRTA-CReSA and Jordi Charles from ble neutralizing humoral response to MERS-CoV (Fig. 2d). IRTA for their technical support. MPSP-RBD vaccination induced high titres of virus nAbs in sera, which were boosted in 2 out of 3 animals three Authors’ contributions J.R., A.B., J.V.-A., B.L.H., J.S. conceived and designed the experiment. J.R., A.Z.M., weeks after contact with MERS-CoV-inoculated llamas G.C., I.C.A, B.-J.B., A.B., J.-C.A., J.V.-A., B.L.H., J.S. performed the experiments shedding high titres of infectious virus (Fig. 2d). Thus, the and analyzed the data. The manuscript was written by J.R. All the authors MPSP-RBD vaccine candidate was able to partially prevent discussed the results and substantively revised the manuscript. The author(s) read and approved the final manuscript. MERS-CoV transmission among camelids, being effective in 1/3 of the animals vaccinated in this exploratory study. Authors’ information Based on the enhanced immune response offered Jordi Rodon is a predoctoral researcher in the Animal Health Research Pro‑ gram (CReSA) of IRTA, Barcelona, Spain. His interests are basic and translational by MPSP-displayed immunogens and the in vivo pro- research in re-emerging viral zoonotic diseases with pandemic potential; his tective capacity of the MPSP-RBD vaccine prototype research is highly devoted to the ‘One Health’ initiative. against MERS-CoV [18], we evaluated its potential to Funding inhibit MERS-CoV transmission among camelid reser- This study was performed as part of the Zoonotic Anticipation and voirs. Immunization with the MPSP-RBD formulated Preparedness Initiative (ZAPI project) [Innovative Medicines initiative (IMI) with a commercial adjuvant elicited nAbs to MERS-CoV grant 115760], with assistance and financial support from IMI and the European Commission and contributions from EFPIA partners. J.R. was but transmission was only prevented in 1/3 of the ani- partially supported by the VetBioNet project (EU Grant Agreement INFRA- mals. Since high MERS-CoV seroprevalence and evi- 2016–1 Nº731014). IRTA is supported by CERCA Programme / Generalitat de dence of reinfection have been found in camelids [22], Catalunya. further studies would be needed to investigate whether Availability of data and materials MPSP-RBD administration can boost sufficient protec- The datasets used and/or analysed during the current study are available from tive immune responses to MERS-CoV and decrease the the corresponding author on reasonable request. transmission rate in previously exposed animals. The monomeric RBD displayed by MPSP may induce lower Declarations protective responses than a prototype shaping a trimeric Ethics approval and consent to participate conformation or the combination with other S subunits, The present study was approved by the Ethical and Animal Welfare Commit‑ as evidenced by the high efficacy of a previous vaccine tee of IRTA (CEEA-IRTA) and by the Ethical Commission of Animal Experimenta‑ tion of the Autonomous Government of Catalonia (file No. CEA-OH/10942/1). candidate using the same adjuvant and route of admin- istration [5]. Nonetheless, the capabilities of MPSP-RBD Rodon et al. One Health Outlook (2022) 4:12 Page 5 of 5 Consent for publication 13. World Health Organization (WHO). WHO Target Product Profiles for MERS- Not applicable. CoV Vaccines. 2017. (http://​www.​who.​int/​bluep​rint/​what/​resea​rch-​devel​ opment/​MERS_​CoV_​TPP_​15052​017.​pdf ). Competing interests 14. Adney RD, Wang L, van Doremalen N, Shi W, Zhang Y, Kong W-P, et al. 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Sabir JSM, Lam TTY, Ahmed MMM, Li L, Shen Y, Abo-Aba SEM, et al. • rapid publication on acceptance Co-circulation of three camel coronavirus species and recombination of • support for research data, including large and complex data types MERS-CoVs in Saudi Arabia. Science. 2016;351(6268):81–4. 11. Adney DR, van Doremalen N, Brown VR, Bushmaker T, Scott D, de Wit E, • gold Open Access which fosters wider collaboration and increased citations et al. Replication and shedding of MERS-CoV in upper respiratory tract of • maximum visibility for your research: over 100M website views per year inoculated dromedary camels. Emerg Infect Dis. 2014;20(12):1999–2005. 12. Haagmans BL, van den Brand JMA, Raj VS, Volz A, Wohlsein P, Smits SL, At BMC, research is always in progress. et al. An orthopoxvirus-based vaccine reduces virus excretion after MERS- CoV infection in dromedary camels. Science. 2016;351(6268):77–81. Learn more biomedcentral.com/submissions