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Veterinary Immunology and Immunopathology 218 (2019) 109939 Contents lists available at ScienceDirect Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm Short communication Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal T tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein Ann-Kathrin Haverkampa,1, Berend J. Boschb,1, Ingo Spitzbartha,c, Annika Lehmbeckera,c, ⁎ Nigeer Ted, Albert Bensaidd, Joaquim Segalése,f, Wolfgang Baumgärtnera,c, a Department of Pathology, University of Veterinary Medicine Hannover Foundation, 30559 Hannover, Germany b Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, the Netherlands c Center for Systems Neuroscience, 30559 Hannover, Germany d IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain e Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, UAB, 08193 Bellaterra, Barcelona, Spain f UAB, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain A R T I C LE I N FO A B S T R A C T Keywords: Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal Immunohistochemistry outcome in one third of human patients. In recent years, several investigators developed protective antibodies Middle East respiratory syndrome coronavirus which could be used as prophylaxis in prospective human epidemics. In the current study, eight human Spike protein monoclonal antibodies (mAbs) with neutralizing and non-neutralizing capabilities, directed against different Monoclonal human antibodies epitopes of the MERS-coronavirus (MERS-CoV) spike (MERS-S) protein, were investigated with regard to their ability to immunohistochemically detect respective epitopes on formalin-fixed paraffin-embedded (FFPE) nasal tissue sections of MERS-CoV experimentally infected alpacas. The most intense immunoreaction was detected using a neutralizing antibody directed against the receptor binding domain S1B of the MERS-S protein, which produced an immunosignal in the cytoplasm of ciliated respiratory epithelium and along the apical membranous region. A similar staining was obtained by two other mAbs which recognize the sialic acid-binding domain and the ectodomain of the membrane fusion subunit S2, respectively. Five mAbs lacked immunoreactivity for MERS- CoV antigen on FFPE tissue, even though they belong, at least in part, to the same epitope group. In summary, three tested human mAbs demonstrated capacity for detection of MERS-CoV antigen on FFPE samples and may be implemented in double or triple immunohistochemical methods. 1. Introduction Even though MERS-CoV is most likely derived from an ancestral reservoir in bats (Corman et al., 2014; Cui et al., 2013; de Wit and Middle East respiratory syndrome coronavirus (MERS-CoV) is a Munster, 2013; Memish et al., 2013; Munster et al., 2016; van novel lineage C betacoronavirus, which was first identified in a patient Boheemen et al., 2012), transmission to humans occurs mainly via from the Kingdom of Saudi Arabia in June 2012 (Zaki et al., 2012). As dromedaries, which represent a major reservoir for the virus in large of May 2019, MERS-CoV has been detected in 27 countries and infected parts of the Middle East, the Canary Islands, Africa, and Southern Asia more than 2300 patients (World Health Organization (2019) Middle (Falzarano et al., 2017; Haagmans et al., 2014; Hemida et al., 2017; East respiratory syndrome coronavirus, available at https://www.who. Saqib et al., 2017). Infection has also been demonstrated in farmed int/csr/don/24-April-2019-mers-saudi-arabia/en/). Although the dis- alpacas from a region of Qatar where MERS-CoV is endemic (Reusken ease vanished almost completely from headlines and newspapers, it is et al., 2016), but not in any other livestock species (Reusken et al., still a potential threat to human health since approximately one third of 2013). Recently, experimental inoculation of pigs, alpacas, and llamas all affected patients succumb to acute respiratory distress syndrome and with MERS-CoV resulted in low levels of viral replication in pigs and renal dysfunction (Zumla et al., 2015). moderate to high levels of viral replication and shedding in the latter ⁎ Corresponding author at: Department of Pathology, University of Veterinary Medicine Hannover Foundation, Bünteweg 17, 30559 Hannover, Germany. E-mail address: Wolfgang.Baumgaertner@tiho-hannover.de (W. Baumgärtner). 1 both authors contributed equally. https://doi.org/10.1016/j.vetimm.2019.109939 Received 10 May 2019; Received in revised form 2 September 2019; Accepted 8 September 2019 0165-2427/ © 2019 Elsevier B.V. All rights reserved. A.-K. Haverkamp, et al. Veterinary Immunology and Immunopathology 218 (2019) 109939 Table 1 Antibodies, clonality, species, antigen retrieval, dilution, secondary antibodies and results. Antibody Clonality, host species (lot number) Protein content [μg/ml] Antigen retrieval Dilution Secondary antibody Result 1.2g5 mc, human 0.23 # 1:5, 1:10, 1:20 GaH-b + 1.10f3 0.27 # 1:5, 1:10, 1:20 (+) 1.6c7 0.27 citrate 1:5 (+) 1.6f9 0.23 1:5 – 4.6e10 0.43 1:5 – 7.7g6 0.77 1:5 – 1.8e5 0.25 1:5 – 3.5g6 0.91 1:5 – MERS-CoV nucleocapsid (Sino Biological) mc, mouse (HB10AP1804-B) 1.0 1:70 GaM-b +++ MERS-CoV nucleocapsid (Sino Biological) pc, rabbit (HB07AP1208) 1.0 1:2000 GaR-b +++ GaH-b, goat anti-human IgG biotinylated; GaM-b, goat anti-mouse IgG biotinylated; GaR-b, goat anti-rabbit IgG biotinylated; mc, monoclonal; MERS-CoV, Middle East respiratory syndrome coronavirus; pc, polyclonal; #, antigen retrieval was performed with boiling citrate buffer, proteinase K and omission of pretreatment; + ++, intense antigen specific signal in several cells, minimal background staining (Fig. 1C, 1D); +, moderately intense antigen specific signal in fewer cells, minimal background staining (Fig. 2A); (+), moderately intense antigen specific signal in fewer cells, strong background staining (Fig. 2C, D); -, no antigen specific signal, varying degrees of background staining. species, respectively (Adney et al., 2016a; de Wit et al., 2017; Vergara- 2. Materials and methods Alert et al., 2017). Similarly, minimal virus shedding was detected in goats, sheep, and horses after experimental infection, but only goats 2.1. Human monoclonal antibodies developed neutralizing antibodies (Adney et al., 2016b). Due to the close interactions of humans with potentially susceptible and con- The development of the eight diverse human mAbs directed against tagious livestock species in certain parts of the world, the development different epitopes of MERS-S protein has been published previously of an effective vaccination strategy against MERS-CoV was proposed (Widjaja et al., 2019). Shortly, generation of anti-MERS-S H2L2 mAbs early after its initial discovery and was recently followed up by the was achieved by recurrent immunization of transgenic H2L2 mice, development of protective antibodies by different protocols (Agrawal which encode the human immunoglobulin variable regions, with the et al., 2016; Corti et al., 2015; Houser et al., 2016; Jiang et al., 2014; respective antigens and adjuvants (for preparation of antigens see ori- Johnson et al., 2016; Li et al., 2015; Pascal et al., 2015; Qiu et al., 2016; ginal publications (Li et al., 2017; Mou et al., 2013; Walls et al., 2016; Tang et al., 2014; Widjaja et al., 2019). Widjaja and colleagues (2019) Widjaja et al., 2019)). At day 74, four days after the last immunization, developed a set of protective neutralizing and non-neutralizing human spleen and lymph nodes were harvested and hybridoma cell lines were monoclonal antibodies (mAbs) which target different epitopes of the generated using the SP2/0 myeloma cell line. For production of re- MERS-CoV spike (MERS-S) protein. The MERS-S protein is known to combinant mAbs, cDNA’s encoding the variable heavy and light chain represent a key target for the development of new therapeutics and regions of anti-MERS-S H2L2 mAbs were cloned into expression plas- consists of a receptor-binding subunit S1 and a membrane-fusion sub- mids containing the human IgG1 heavy chain and Ig kappa light chain unit S2 (Du et al., 2017). The subunit S1 is, in turn, composed of four constant regions, respectively. Recombinant human anti-MERS-S mAbs different core domains. The domain S1B binds to the host-cell receptor were produced in HEK-293 T cells following co-transfection of the IgG1 dipeptidylpeptidase 4 (DPP4; Lu et al., 2013; Raj et al., 2013; Wang heavy and light chain expression plasmids (Invivogen) according to et al., 2013), while the domain S1A binds to sialoglycans which en- manufacturers’ protocols (Widjaja et al., 2019). Antibodies were pur- hances infection of human lung cells by MERS-CoV (Li et al., 2017). The ified from tissue culture supernatants using Protein-A affinity chroma- subunit S2 comprises the fusion peptide, two heptad repeats and a tography. Purified antibodies were quantified using UV absorbance transmembrane domain, which mediate fusion of the virus with the cell (280 nm) and Coomassie stained SDS-PAGE analysis was applied in membrane (Lu et al., 2013; Pallesen et al., 2017; Raj et al., 2013; Wang addition. Antibodies were stored at 4 °C until further use. After et al., 2013). screening for MERS-S reactivity and neutralizing capacity eight dif- In the present study, these mAbs were tested for im- ferent mAbs (Table 1) were selected based on epitope distribution and munohistochemistry to detect respective epitopes on formalin-fixed neutralization. paraffin-embedded (FFPE) nasal tissue sections of MERS-CoV infected alpacas. Immunohistochemistry is a well-established and powerful method which is frequently used for diagnostic purposes and evaluation 2.2. Tissue of animal experiments. Simple protocols allow precise correlation be- tween the presence and severity of lesions and the amount of viral Formalin-fixed tissue of nasal turbinates from two experimentally antigen in the respective localization (Duraiyan et al., 2012). Even infected alpacas was obtained from an animal trial recently im- though there are several mono- and polyclonal antibodies available for plemented at the Research Center for Animal Health (Centre de Recerca the detection of the MERS-CoV nucleocapsid and spike protein on FFPE en Sanitat Animal, CReSA, Barcelona, Spain). The mucosa was carefully tissue (Haverkamp et al., 2018), these antibodies are all derived from removed from underlying bone and embedded in paraffin according to rabbits and mice, respectively (https://www.biocompare.com, accessed a routinely used protocol. Serial sections of 4 μm thickness were March 11, 2019). However, for double or triple immunohistochemistry mounted on coated glass slides (Superfrost Plus®, Menzel Co.) and the and -fluoresence labeling, it can be useful to have access to epitope- first section was stained with HE for evaluation by light microscopy and specific antibodies from different species, which can be used to visua- subsequent sections were processed for immunohistochemistry. For lize viral antigen in tissue sections. It was therefore the aim of the negative control, nasal turbinate of a non-infected alpaca (collected present study to evaluate the suitability of different human anti-MERS-S during routine necropsy at the Department of Pathology, University of mAbs for detection of the respective antigen on FFPE material. Veterinary Medicine Hannover, Germany) was used. The animal died due to cachexia related to non-respiratory disease. 2 A.-K. Haverkamp, et al. Veterinary Immunology and Immunopathology 218 (2019) 109939 2.3. Immunohistochemistry antigen retrieval showed the strongest signal of all tested non-com- mercially available antibodies. Detected antigen was present multi- Sections were dewaxed in Roticlear® (Roth C. GmbH & Co. KG) and focally in the cytoplasm of ciliated respiratory epithelium and seg- subsequently rehydrated in isopropanol and 96% ethanol (Roth C. mentally along the apical membranous region of few infected cells GmbH & Co. KG). Endogenous peroxidase activity was blocked by in- (Fig. 2A). The observed staining pattern was comparable to descriptions cubation of sections in 85% ethanol with 0.5% H2O2 (VWRTM by others investigating the distribution of MERS-CoV in tissue sections International GmbH) for 30 min at room temperature and antigen re- of different infected animal species (Adney et al., 2014; Haagmans trieval was performed by incubation in citrate buffer (2.1 g citric acid et al., 2016; Haverkamp et al., 2018; Vergara-Alert et al., 2017) but the monohydrate in 1 l distilled water, adjusted with NaOH to pH = 6.0) number of labeled cells was considerably lower. Staining with the same for 20 min in a microwave (800 W). In a preliminary experiment two of antibody without pretreatment revealed a similar distribution of viral the eight antibodies (1.10f3 and 1.2g5) were additionally tested antigen in a comparable number of cells but a less prominent expression without pretreatment and with enzymatic digestion by Proteinase K pattern (Fig. 2B), which is most likely linked to the lack of antigen antigen retrieval solution (Merck KGaA) according to the manu- unmasking which takes place during antigen retrieval (Shi et al., 2011). facturer’s protocol. All sections were transferred to Shandon Detection of MERS-CoV antigen by the 1.2g5 antibody was almost Coverplates™ (Thermo Electron GmbH) and nonspecific binding was completely absent with pretreatment by enzymatic digestion in all three blocked by inactivated 20% goat serum diluted in phosphate buffered dilutions, resulting only in a pale brownish signal interpreted as non- saline (PBS) including 1% bovine serum albumin (BSA; PBS/BSA) for specific background staining (data not shown). The antibody 1.10f3 30 min. Afterwards sections were incubated with the primary antibody showed a strong non-specific background staining in all dilutions and (1.2g5, 1.6c7, 1.10f3, 1.6f9, 4.6e10, 7.7g6, 3.5g6, and 1.8e5) in their pretreatments. A moderately intense antigen-specific signal was de- respective dilution (Table 1) for 90 min at room temperature and the tected multifocally in cilia of few cells using the lowest dilution and secondary biotinylated antibody diluted in PBS (1:200) was added. citrate pretreatment (Fig. 2C). Incubation for 60 min at room temperature was followed by treatment Of the six remaining antibodies (1.6c7, 1.6f9, 4.6e10, 7.7g6, 3.5g6, with the avidin-biotin-peroxidase complex (Vectastain ABC Kit Stan- 1.8e5), only antibody 1.6c7 showed a mild multifocal staining of cilia dard, VectorLaboratories) according to the manufacturer’s protocol. accompanied by a strong background reactivity (Fig. 2D). The other Visualization of the reaction was achieved by addition of chromogen five antibodies (1.6f9, 4.6e10, 7.7g6, 1.8e5, 3.5g6) did not reveal any 3,3-diaminobenzidine tetrahydrochloride (DAB, 0.05%, Sigma Aldrich recognizable specific staining of MERS-CoV antigen on FFPE tissue Chemie GmbH) and 0.03% H2O2. Sections were finally slightly coun- along with no to very mild (1.6f9, 4.6e10, Fig. 3A, B), mild (7.7g6, terstained with Mayer’s hematoxylin (Roth C. GmbH & Co KG). For 1.8e5, Fig. 3C, D) and strong (3.5g6, Fig. 3E) non-specific background positive control and elucidation of virus distribution, additional sec- staining. By contrast, all antibodies displayed a distinct signal by im- tions were stained with two different commercially available, pre- munofluorescence staining of MERS-CoV-infected Huh-7 cells in culture viously published monoclonal mouse and polyclonal rabbit anti-MERS- (Widjaja et al., 2019). Possible explanations for the lack of im- CoV nucleocapsid antibodies (Sino Biological Inc.; Haagmans et al., munoreactivity on FFPE tissue include effacement of conformational 2016; Haverkamp et al., 2018), respectively. For negative controls, the epitopes by formalin fixation and/or antigen retrieval protocols primary antibody was replaced by ascites fluid from Balb/c mice (Smedley et al., 2007). (1:1,000) and normal rabbit serum (1:3,000), respectively. Moreover, The tested antibodies in the present study were derived from all antibodies were applied to tissue of a non-infected alpaca (section transgenic mice expressing chimeric antibodies containing human 2.2). variable domains and constant antibody domains of rat origin. For generation of fully human antibodies the human variable heavy and 3. Results and discussion light chain encoding gene segments of the antibodies isolated from MERS-S immunized mice transgenic for human V genes were cloned Evaluation of HE stained slides by light microscopy revealed a mild into human IgG1 heavy and light chain expression vectors, respectively to moderate, multifocal, lymphoplasmahistiocytic and suppurative (Widjaja et al., 2019). All these antibodies are directed against MERS-S rhinitis characterized by low numbers of intraepithelial neutrophils and and belong to six distinct epitope groups that interfere with three cri- lymphocytes and infiltration of the lamina propria and submucosa by tical entry functions in vivo. In particular, 1.2g5, 7.7g6, 1.6f9, 1.8e5, low to moderate numbers of lymphocytes, single plasma cells, and and 4.6e10 are neutralizing antibodies directed against the receptor macrophages (Fig. 1A, B). These findings are reminiscent of previous binding domain (RBD) S1B of the MERS-S protein (Widjaja et al., 2019), observations in other large animal species though lesions in alpacas are which facilitates virus binding to the DPP4 receptor on the host cell relatively mild and lack the necrotizing component that has been de- surface (Mou et al., 2013; Raj et al., 2013). The neutralizing antibodies scribed for dromedaries, llamas, and pigs (Adney et al., 2014; 1.6c7 and 3.5g6 bind to the ectodomain of the membrane fusion sub- Haagmans et al., 2016; Vergara-Alert et al., 2017). The commercially unit S2 which mediates fusion of the viral and cellular membranes (Li available monoclonal and polyclonal anti-MERS-CoV nucleocapsid an- et al., 2017; Widjaja et al., 2019). By contrast, antibody 1.10f3 is a non- tibodies, which were used as positive controls, revealed a strong in- neutralizing antibody that recognizes the sialic acid binding domain tracytoplasmic and cilia-associated staining pattern in numerous cells (Widjaja et al., 2019). Since the positive staining antibodies 1.2g5, (Fig. 1C, D) according to formerly published results (Haagmans et al., 1.10f3 and 1.6c7 recognize epitopes that belong to different functional 2016; Haverkamp et al., 2018). A specific staining was absent in all groups (DPP4 binding, sialic acid binding and membrane fusion, re- negative controls as expected. spectively), the suitability of mAbs for use in immunohistochemistry For evaluation of human mAbs by immunohistochemistry, two of cannot be predicted from the function of their target epitopes in vivo. the tested antibodies (1.2g5 and 1.10f3) were applied in three different dilutions and three different pretreatments in a preliminary experiment 4. Conclusion to identify the best method for the use of human mAbs on FFPE tissue (Table 1). As a result, heat-induced antigen retrieval and application of The present study shows that three tested human mAbs, designated the antibody in the lowest dilution revealed the best staining effects. 1.2g5, 1.10f3, and 1.6c7, are suitable to detect MERS-CoV antigen on According to these results, all other antibodies (1.6c7, 1.6f9, 4.6e10, FFPE tissue sections of experimentally infected alpacas, though to a 7.7g6, 3.5g6, 1.8e5) were tested with heat-induced antigen retrieval limited extend. The best immunolabelling results for all three anti- and in the lowest dilution. bodies were achieved at the lowest dilution (1:5) with heat-induced The use of the 1.2g5 antibody in a 1:5 dilution with heat-induced antigen retrieval. However, results demonstrate that the suitability of 3 A.-K. Haverkamp, et al. Veterinary Immunology and Immunopathology 218 (2019) 109939 Fig. 1. Representative HE findings in respiratory epithelium of the nasal tur- binates of MERS-CoV-infected alpacas and immunohistochemical reactions of the commercially available positive controls for comparison. (A) Multifocal infiltration of lamina propria and sub- mucosa by moderate numbers of lym- phocytes, macrophages, and single neutrophilic granulocytes (asterisk). (B) Exocytosis of single neutrophilic gran- ulocytes (grey arrow). (C, D) Abundant viral antigen was detected multifocally in the cytoplasm and along the apical membranous region of epithelial cells using a monoclonal mouse (C, Sino Biological Inc.) and polyclonal rabbit anti-MERS-CoV nucleocapsid antibody (D, Sino Biological Inc.) with citrate pretreatment, in a dilution of 1:70 and 1:2,000, respectively. Both antibodies exhibited a similar staining intensity. (A, B) HE staining; 400x, (C, D) Avidin- biotin-peroxidase complex method with 3,3′-diaminobenzidine as chromogen; 400x. Fig. 2. Representative positive im- munohistochemical reactions for human monoclonal antibodies. (A) Antibody 1.2g5 with citrate pretreat- ment exhibited a strong multifocal MERS-CoV antigen specific signal in the cytoplasm of ciliated respiratory epi- thelial cells (arrow) and segmentally along the apical membranous region (arrow heads). (B) The same protocol without pretreatment revealed a much fainter but similarly distributed staining of cytoplasm (arrow) and ciliary base (arrow heads). (C) Antibody 1.10f3 with citrate pretreatment displayed a strong diffuse background staining and only few positively stained cilia (ar- rowhead). Single intraepithelial plasma cells displayed a strong false positive intracytoplasmic staining (white arrow). (D) Antibody 1.6c7 with citrate pretreatment exhibited also a strong diffuse background staining and only few positive staining cilia (arrow heads). (A–D) Avidin-biotin-peroxidase complex method with 3,3′-diamino- benzidine as chromogen; 400x. the antibodies was unrelated to the function of their target epitopes in human mAbs might represent a promising alternative. vivo and that commercially available monoclonal and polyclonal anti- nucleocapsid antibodies generally appear more suitable to detect MERS-CoV antigen on FFPE sections in routine diagnostics. However, Acknowledgements for certain purposes such as double or triple immunohistochemistry, The authors would like to thank Petra Grünig, Caroline Schütz, 4 A.-K. Haverkamp, et al. Veterinary Immunology and Immunopathology 218 (2019) 109939 Fig. 3. Representative negative immunohistochemical reactions for human monoclonal antibodies. (A–E) Specific staining for MERS-CoV antigen was absent for the antibodies 1.6f9 (A), 4.6e10 (B), 7.7g6 (C), 1.8e5 (D), and 3.5g6 (E), respectively. Reactions were accompanied by mild to severe non-specific, intracytoplasmic background staining. 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