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Article

Construction and Application of a Canine SLAM Receptor-Based System from Vero Cell Line to Virus Isolation and Parallel Antibody Screening

by
Jianbo Xu
1,
Xiangda Hu
1,
Sizhuo Chen
2,
Zhenhuan Zhao
2,
Zhangchang Wang
3,
Minghui Wang
4,
Zhongchuan Tang
4,
Mingqian Feng
1,
Zhiyuan Zhao
3,* and
Xin Chen
1,2,*
1
College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
2
Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan 430068, China
3
Wuhan Academy of Agriculture Science, Wuhan 430070, China
4
Huanggang Academy of Agricultural Sciences, Huanggang 438000, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2026, 27(4), 1895; https://doi.org/10.3390/ijms27041895
Submission received: 5 January 2026 / Revised: 11 February 2026 / Accepted: 12 February 2026 / Published: 16 February 2026
(This article belongs to the Special Issue Antiviral Drug Targets: Structure, Function, and Drug Design—3rd Edition)
Browse Figures
Versions Notes

Abstract

Canine distemper virus (CDV) remains a highly contagious and lethal pathogen, posing a severe global threat to domestic dogs and wild carnivores. To address the urgent need for effective interventions, we utilized a proprietary Vero-SLAM cell platform to isolate a wild-type CDV strain and generate neutralizing polyclonal antibodies. Subsequently, phage display technology was employed to screen for single-chain variable fragments (scFvs) targeting the CDV hemagglutinin protein (CDV-H). This approach led to the identification of a specific scFv with virus-binding affinity comparable to commercial antibodies, which effectively blocks CDV infection in Vero-SLAM cells. Molecular docking and molecular dynamics simulations were conducted to elucidate the interaction mechanism, suggesting that this scFv binds to a novel and unique epitope on the CDV-H. These findings not only expand our understanding of the antigenic properties of the CDV H protein but also provide a theoretical foundation and a promising candidate molecule for the development of future CDV diagnostics and antiviral strategies.

1. Introduction

Canine distemper virus (CDV), a member of the genus Morbillivirus within the family Paramyxoviridae, is an enveloped, non-segmented negative-strand RNA virus primarily transmitted via the respiratory route in canines, causing canine distemper (CD). Its clinical manifestations resemble those of human measles virus infection, encompassing high fever, rash, diarrhea, mucopurulent nasal discharge, and profound immunosuppression, often accompanied by acute or delayed neurological complications such as encephalitis and locomotor deficits [1,2]. The host range of CDV has expanded beyond traditional Canidae to encompass all eight families within the order Carnivora, including fur-bearing species such as foxes, minks, and ferrets, with sporadic detections in Suidae (Artiodactyla) and Macaca (Primates), resulting in substantial economic losses to wildlife conservation, fur farming, and the pet industry [3,4,5,6]. In China, the rapid expansion of livestock and pet economies has elevated CDV outbreaks as a primary threat to breeders, underscoring the urgent need for effective control measures.
Since its identification in 1905, various live-attenuated vaccines have been developed [7]. However, waning immunity and the emergence of novel variants increase the risk of vaccine failure [8,9]. The high genetic variability of the H gene, the most divergent region in the CDV genome, is a primary cause of vaccine failure, resulting in reduced efficacy of existing vaccines against wild-type CDV strains [10]. The genetic diversity of CDV is reflected in its H gene, which forms the basis for categorization into seven distinct geographic genotypes: America-1, America-2, Asia-1, Asia-2, Europe, Arctic-like, and European wildlife [11,12]. Subsequent studies identified additional genotypes such as South America-2, Mexico, and Argentina [13,14]. The Asia-1 genotype predominates in China, consistent with experimental isolates. Continued research reveals that CDV’s genetic diversity exceeds that of related morbilliviruses like measles virus (MeV) and rinderpest virus (RPV), with a broader host range [6]. Although vaccination has reduced large-scale outbreaks in dogs, cross-species transmission remains frequent. Given CDV’s host expansion and similarity to MeV, reported primate infections have raised concerns about potential human transmission [15,16,17]. Bieringer et al. demonstrated that mutated CDV H protein could infect human signaling lymphocytic activation molecule (SLAM)expressing cells, indicating a potential risk under specific conditions [18]. While no human infections have been documented, likely due to widespread MeV vaccination, the potential zoonotic threat warrants vigilance.
CDV exists as a single serotype, though strains exhibit significant differences in pathogenicity, plaque morphology, and neurovirulence, despite similarities in structural proteins and genomic organization. As an enveloped virus, CDV has low environmental resistance and is sensitive to temperature, leading to seasonal peaks in spring and winter [19]. The H and fusion (F) glycoproteins are major immunogens and key determinants of infectivity, frequently associated with vaccine failures. Vaccine strains like Onderstepoort utilize CD46 as a receptor and can be cultured in Vero cells, whereas wild-type strains require SLAM- or nectin-4-expressing cells, reflecting their tropism for lymphoid, epithelial, and neural cells [1,20,21,22]. The H protein readily induces immune responses, making it a common component of genetic vaccines [23]. The F protein cooperates with H to mediate membrane fusion; Meertens et al. demonstrated its role in promoting non-cytolytic cell-to-cell spread during persistent infection [24]. CDV vaccine strains typically cause non-lytic infections, whereas wild-type strains induce cell fusion and cytopathic effects.
This study established a Vero-SLAM platform and a CDV immune library, enabling the isolation of wild-type virus and the identification of the H protein-targeting CDV-R01. Computational simulations revealed the scFv’s binding mechanism to CDV-H and identified key CDR residues that underpin its potential neutralizing function.

2. Results

2.1. Development of a Canine SLAM-Positive Vero Cell Platform for CDV Research

Recombinant canine SLAM extracellular domain (ECD) fused to human Fc was transiently expressed in Expi-293F cells and purified to >90% homogeneity, As shown in Figure 1A, SDS-PAGE analysis revealed a distinct band at approximately 150 kDa under non-reducing (non-re) conditions, corresponding to the disulfide-linked homodimer. Under reducing (re) conditions, the protein migrated as a single band at approximately 70 kDa. Although the theoretical molecular weight of the SLAM (ECD)-hFc monomer is calculated to be ~50 kDa, the observed migration at 70 kDa suggests the presence of post-translational modifications, such as glycosylation, which are characteristic of proteins expressed in mammalian systems. This purified SLAM (ECD)-hFc protein (hFc: human IgG Fc fragment) was used to immunize chicken C037, yielding polyclonal antiserum with high antigen-binding titers against SLAM ECD-hFc, as determined by ELISA (Figure 1B). Titration curves demonstrated robust binding (EC50 = 6400 dilution), with negligible reactivity to unrelated IAB-hFc antigen, confirming specificity.
Full-length canine SLAM cDNA was transfected into parental Vero cells, followed by puromycin selection over multiple rounds to establish stable transfectants. Monoclonal Vero-SLAM clones were further enriched by fluorescence-activated cell sorting (FACS) using PE-conjugated anti-SLAM antibody. Surface SLAM expression was validated by flow cytometry, revealing a marked shift in PE-A fluorescence intensity in SLAM-stained Vero-SLAM clones (specimens 004) compared to unstained (green) or mock-stained (blue) controls (Figure 1C). Geometric mean fluorescence intensities (Geo Mean PE-A) were substantially elevated in sorted populations (e.g., 4138 for specimen 004 vs. 29.7 for specimen 001unstained), indicating successful enrichment of high-SLAM expressors. Complementary qRT-PCR analysis confirmed expression of SLAM mRNA in puromycin-selected Vero-SLAM cells relative to parental Vero cells (Figure 1D; n = 3; mean ± SD).
The functionality of Vero-SLAM cells as a CDV propagation platform was assessed by infection with wild-type CDV. Infection of Vero-SLAM cells induced pronounced cytopathic effects (CPEs), including syncytium formation and plaque lysis, which were absent in parental Vero cells (Supplementary Figure S1A). Leveraging this enhanced susceptibility, we successfully isolated three wild-type CDV strains from field samples: CDV-Shamo, CDV-Hashiqi, CDV-Jinmao (Supplementary Figure S1B). These Vero-SLAM cells thus provide a robust, receptor-authenticated platform for CDV research, enabling efficient virus isolation and genetic characterization.

2.2. Development of a Phage-Displayed scFv Library Against CDV

New Zealand White rabbits (R064 and R065) were immunized with 108 PFU of the highly virulent CDV-Shamo strain, administered over four boosts. Serum samples collected two days after each immunization were evaluated for antigen-binding titers by ELISA against CDV-Shamo antigen. Post-immune sera from both rabbits exhibited robust titers after the fourth immunization, with significant increases compared to pre-immune baselines (Figure 2A,B). Titration curves revealed EC50 values ranging from approximately 1:1600 to 1:5000 for post-immune sera, demonstrating the induction of high-titer antibodies specific to CDV antigens.
Neutralizing activity of the post-fourth immunization sera, alongside pre-immune controls, was assessed via plaque reduction neutralization assay on Vero-SLAM cells challenged with each of the three isolated CDV strains (Jinmao, Shamo, Hashiqi). R064 serum potently inhibited plaque formation across all strains, resulting in near-complete reduction of cytopathic effects and visible plaques upon crystal violet staining (Figure 2C). In contrast, R065 serum displayed moderate neutralization, while naive sera showed no inhibitory effect, highlighting the significantly higher neutralizing efficacy of R064.
To derive a targeted antibody repertoire, total RNA was extracted from the spleen of rabbit R064 and reverse-transcribed to cDNA. Variable light (VL) and heavy (VH) chain genes were amplified by PCR, yielding amplicons of ~350 bp and ~400 bp, respectively, as verified by agarose gel electrophoresis (Figure 2D, upper panels). These were assembled into single-chain variable fragments (scFv) via overlap extension PCR using a flexible (GGGGS)n linker, followed by cloning into the phagemid vector pComb3X. The resulting scFv-pComb3X constructs (~3500 bp) were confirmed by gel electrophoresis (Figure 2D, lower panel) and electroporated into electrocompetent E. coli TG1 cells to generate a phage-displayed scFv library, estimated at >107 transformants (Figure 2D, schematic).
The library underwent three rounds of biopanning by ELISA against immobilized CDV antigens to enrich for antigen-specific binders. From the resulting pool, a high-affinity scFv clone was selected for further characterization based on its robust CDV binding profile (OD450 > 2.0 at a 1:100 dilution) and selective reactivity over irrelevant controls.

2.3. Expression and Binding Activity of the Anti-CDV scFv

The anti-CDV scFv clone (designated CDV-R01) isolated from the phage-display library was genetically fused at its C-terminus to the human Fc domain (hFc) to facilitate secretion, purification, and detection. SDS-PAGE analysis was performed to verify the molecular weight and purity of the protein (Figure 3A). Under non-reducing conditions, the protein migrated as a predominant band of approximately 110 kDa, consistent with the expected molecular weight of the disulfide-linked dimeric fusion protein. Under reducing conditions, a major band was observed at approximately 55 kDa, corresponding to the monomeric scFv-Fc fusion. This analysis confirmed that the purified protein maintained the correct oligomeric state with a purity exceeding 90%.
The binding specificity and affinity of purified CDV-R01 were evaluated using dose-dependent ELISA against immobilized cell lysates from three isolated CDV strains (Shamo, Jinmao, Hashiqi) and a vaccine strain. CDV-R01 exhibited concentration-dependent binding to all tested strains, with half-maximal effective concentration (EC50) values ranging from 0.5 to 1.2 μg/mL (Figure 3B). The strongest binding affinity was observed against the Shamo strain (EC50 = 0.5 μg/mL), which was used as the immunogen. Negligible binding signals (<0.1 OD450) were detected in irrelevant control samples, confirming the high specificity of CDV-R01 for CDV antigens.
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded lung sections from CDV-infected and non-infected dogs to evaluate the recognition of CDV in pathological tissues. CDV-R01 specifically labeled viral inclusions and infected alveolar epithelia in CDV-positive lung tissues, showing intense brown cytoplasmic and membranous staining at 400× magnification (Figure 3C, right panels). In contrast, no specific staining was observed in negative control lung sections (Figure 3C, left panels), demonstrating the utility of CDV-R01 for specific detection of CDV in histopathological contexts without cross-reactivity to host tissues.
Western blot analysis further confirmed the ability of CDV-R01 to recognize native CDV proteins. When cell lysates from three wild-type strains and one vaccine strain were probed, a prominent immunoreactive band of approximately 70 kDa was detected in all samples, corresponding to the CDV hemagglutinin glycoprotein (Figure 3D). Neutralization assays demonstrated that CDV-R01 could significantly reduce viral infection efficiency (partial inhibition) in Vero-SLAM cells, although it did not achieve complete neutralization under the tested conditions (Figure 3E).

2.4. Exploring the CDV-R01-CDV-H Interaction Mechanism by Molecular Docking

The three-dimensional structure of the CDV-R01 variable domain was predicted using AlphaFold3, achieving a predicted TM-score (pTM) of 0.9. Confidence assessment via pLDDT scores highlighted regions of high structural variability, particularly in the CDR-H3 and CDR-L3 loops, which are critical for antigen recognition (Figure 4A). The stereochemical quality of the scFv model was validated using a Ramachandran plot, demonstrating robust backbone geometry: among 186 non-glycine and non-proline residues, 169 (90.9%) occupied most favored regions, 15 (8.1%) additional allowed regions, 0 (0.0%) generously allowed regions, and 2 (1.1%) disallowed regions (predominantly terminal residues), with all residues falling within acceptable conformational space (Figure 4B).
The hemagglutinin protein extracellular domain of the CDV-Shamo strain, comprising 472 residues, was constructed via homology modeling using chain C of PDB entry 7ZNY as the template. Ramachandran plot analysis confirmed the model’s high stereochemical quality, rendering it appropriate for subsequent docking simulations (Supplementary Figure S2). To elucidate the interaction interface, molecular docking was conducted using HADDOCK 2.4 (https://wenmr.science.uu.nl/haddock2.4, accessed on 10 January 2025) designating residues within the scFv CDR-H3 and CDR-L3 loops as active sites for CDV-H engagement, alongside key residues previously identified in CDV-H/SLAM interactions [22]. From 170 generated water-refined models, HADDOCK identified 7 clusters encompassing 85% of the poses. The highest-ranking cluster (cluster 2; Z-score = −1.5) was selected for in-depth examination, with the optimal pose (cluster 2-1) disclosing an expansive binding interface between CDV-H and CDV-R01, stabilized by numerous hydrogen bonds (Figure 4C). Characteristics of the top 7 clusters, including HADDOCK scores (wherein lower values indicate superior poses), are detailed in Table 1.

2.5. Molecular Recognition Mechanism Revealed by Molecular Dynamics Simulations

To further probe the dynamic stability and binding affinity of the CDV-H/scFv complex, 100 ns molecular dynamics (MD) simulations were performed using GROMACS on the top-ranked HADDOCK pose (cluster 2-1), with three independent replicates conducted for robustness. Analysis of the MD trajectories revealed that the RMSD (Root mean square deviation) profiles for the CDV-H/CDV-R01 complex (red) and isolated CDV-H (blue) exhibited comparable fluctuations, slightly higher than those of the isolated scFv (green), with values stabilizing between 0.2–0.35 nm throughout the simulation and converging around 0.3 nm by the endpoint. Notably, the scFv component within the complex displayed even lower RMSD variability (0.2–0.27 nm), underscoring the overall conformational rigidity of the assembly (Figure 5A).
Evaluation of hydrogen bond (H-bond) formation across the 100 ns trajectories demonstrated sustained intermolecular contacts, with an average of 19.6 H-bonds persisting between CDV-H and CDV-R01 (cutoff ≤ 0.35 nm), alongside consistent close residue pairs (≤0.35 nm), indicative of robust interface maintenance (Figure 5B). Complementary MM/GBSA calculations on the equilibrated final 10 ns segments yielded a polar electrostatic contribution (ΔEEL) of −88.93 kcal/mol, reinforcing the pivotal role of H-bonds in interfacial stabilization (Table 2). The overall binding free energy (ΔGtotal) was estimated at −137.32 kcal/mol, signifying a thermodynamically favorable interaction.
The free energy landscape (FEL), projected onto RMSD and radius of gyration (Rg) coordinates, identified a distinct global minimum, from which the representative low-energy structure was extracted and superimposed onto the initial docking model (RMSD = 1.524 Å over 710 residues), affirming close conformational fidelity between docked and dynamically refined poses (Figure 5C). This superposition further highlighted enhanced compactness at the interface in the MD-refined structure relative to the docking model, with CDV-H and CDV-R01 adopting a more intimately engaged orientation, as visualized in the full trajectory animation (Supplementary Figure S3).
Examination of the FEL minimum-energy conformation disclosed 18 intermolecular H-bonds and 6 salt bridges bridging CDV-H (e.g., T546, Q498, Y527, S503) and scFv (e.g., Y31, R84, Y548, D531), collectively fortifying the binding pocket (Figure 5D; Table 3). Per-residue free energy decomposition via MM/GBSA using gmx_MMPBSA v1.6.3 (https://valdes-tresanco-ms.github.io/gmx_MMPBSA/dev/installation, accessed on 13 October 2025) across the 100 ns simulation further corroborated these findings, with the majority of interface-forming residues exhibiting substantial negative ΔG contributions, thereby validating their critical contributions to complex stability (Figure 5E)

3. Discussion

The CDV-H serves as a pivotal epitope that elicits host-specific cytotoxic T lymphocyte responses and promotes the production of substantial neutralizing antibodies, thereby representing a prime target for novel vaccine development and therapeutic neutralizing antibodies [25]. The CDV-H initiates viral entry by specifically binding to the signaling lymphocyte activation molecule (SLAM) receptor, mediating viral attachment to the host cell membrane and triggering the infection process [26,27]. Consequently, SLAM-overexpressing cell lines, such as the Vero-SLAM system, significantly enhance the isolation efficiency of wild-type CDV strains, effectively overcoming issues of viral attenuation or isolation failure commonly encountered in conventional Vero cell cultures [27,28]. Furthermore, given the high variability of the CDV-H that often leads to vaccine breakthrough, the Vero-SLAM platform provides a critical tool for rapid isolation of emerging variants, supporting the development of updated vaccines and the screening of neutralizing antibodies.
Building upon the established Vero-SLAM platform, we isolated canine distemper virus (CDV) strains and constructed a CDV-specific immune repertoire library (Figure 2) to enable high-throughput screening of single-chain variable fragments (scFvs) targeting the hemagglutinin protein (CDV-H), culminating in the identification of CDV-R01.
To explore the molecular mechanism underlying the neutralizing activity of scFv CDV-R01, we employed an integrated computational approach—combining AlphaFold3 de novo prediction, homology modeling using MODELLER 10.6 (https://salilab.org/modeller/download_installation.htm, accessed on 13 October 2025), molecular docking, and molecular dynamics (MD) simulations—to predict its interaction mode with the CDV-H (Figure 4C and Figure 5). Docking analysis using HADDOCK 2.4 revealed that the CDV-R01 engages the SLAM-binding motif on CDV-H through a broad interface (buried surface area ~2650 Å2), stabilized by extensive van der Waals forces and a hydrogen-bond network (Table 1, Figure 4C). Subsequent MD simulations refined the model, demonstrating complex stability over a 100 ns trajectory (RMSD ~0.3 nm) with an average of 19.6 hydrogen bonds maintained (Figure 5A,B). The minimal conformational deviation (RMSD = 1.524 Å) between the free energy landscape (FEL) minimum-energy structure and the docked model confirmed dynamic stability (Figure 5C). MM/GBSA calculations yielded a total binding free energy of −137.32 kcal/mol, primarily driven by electrostatic and van der Waals interactions (Table 2). Per-residue free energy decomposition identified key binding hotspots: on CDV-H, critical residues (G190, A191, T192, T193, S194, L460, I494, D501, R529, G530, V535, Y539, T544, I545, S546, Y547, T548, P550, R552) largely overlapped with the known SLAM-binding site; on the CDV-R01, key contributions originated from the CDR-H3 (V96, G97, Y98, N99, T100) and CDR-L3 (Y222, S224) loops (Figure 5D,E; Table 3). These computational results not only support the initial docking predictions but also suggest specific residues likely critical for binding, showing a pattern consistent with antigenic epitope studies on the measles virus H protein [29,30].
Based on these findings, CDV-R01 exhibits significant potential for both diagnostic and therapeutic applications. In terms of diagnostics, the high specificity of CDV-R01 demonstrated in our study—evidenced by its ability to distinguish CDV antigens in Western blots (Figure 3D) and specifically stain viral inclusions in infected lung tissues (Figure 3C)—suggests its utility in developing rapid antigen detection kits. Unlike polyclonal antibodies, which may suffer from batch-to-batch variation, the recombinant nature of scFv ensures consistency, making it an ideal candidate for immunochromatographic strips or ELISA-based assays for clinical diagnosis [31]. Therapeutically, the ability of CDV-R01 to block viral infection in vitro (Figure 3E) indicates it could serve as a passive immunotherapy agent. Notably, our characterization confirmed that CDV-R01 specifically binds the viral Hemagglutinin (H) protein (Figure 3D). Since the H protein is responsible for receptor binding and our neutralization assays involved pre-incubating the antibody with the virus, the observed inhibition implies that CDV-R01 primarily acts by blocking viral attachment. This mechanism is further supported by our computational prediction, which shows that CDV-R01 sterically hinders the SLAM-binding site. By targeting the receptor-binding site on the H protein, CDV-R01 may physically prevent viral attachment, offering an alternative intervention for unvaccinated or immunocompromised dogs during outbreaks [32].
Nevertheless, the translation of scFv-based agents into clinical practice faces specific challenges that must be addressed. A primary limitation of native scFv molecules is their small size (~25–30 kDa), which falls below the renal filtration threshold, leading to rapid clearance from the blood and a short in vivo half-life [33,34]. While we engineered an scFv-Fc fusion protein in this study to facilitate purification and detection, native scFvs also generally exhibit lower thermal stability compared to full-length IgGs. Therefore, future work towards in vivo validation should focus on antibody engineering strategies. These include converting the scFv into a full-length IgG format or a bivalent “minibody” to enhance stability and avidity, or employing PEGylation to retard renal clearance [34,35]. Recent studies have highlighted the structural determinants of high-potency antibodies against CDV. Shi et al. reported that a whole-canine IgG (D16) elicited robust therapeutic protection in vivo [32], whereas Xiao et al. demonstrated that while fusing a nanobody to an Fc domain significantly increased its activity (up to 4.6-fold), maximal potency still relied heavily on the intrinsic affinity of the variable domains [25]. The comparison between our scFv-Fc (partial inhibition) and the whole IgG D16 (robust protection) suggests that the scFv-Fc format may lack the rigid structural stability of the native Fab arm found in full-length IgGs, or that the intrinsic affinity of the current scFv clone requires further optimization. Consequently, future work should focus on converting CDV-R01 into a full-length canine IgG structure to mimic the stabilized architecture of therapeutic antibodies, or performing structure-guided mutagenesis—as supported by our computational docking data—to achieve neutralization titers comparable to these leading candidates.
In addition to these intrinsic limitations of scFv molecules, this study has certain experimental limitations. We have not yet quantitatively determined the binding affinity between CDV-R01 and CDV-H using biophysical methods such as surface plasmon resonance (SPR). These experimental validations are essential to fully corroborate the computational predictions. Although our neutralization assays showed partial inhibition, systematic in vivo experiments using ferret or dog models are strictly required to evaluate the biodistribution and protective efficacy against lethal challenge. Additionally, as the current analysis primarily relies on a single strain (Shamo), future work should expand to include globally circulating CDV lineages to comprehensively evaluate the impact of viral sequence diversity on key antigenic epitopes.
In summary, by integrating the Vero-SLAM cell platform, CDV immune library, and multi-scale computational simulations, this study systematically proposes a structural basis for the interaction mechanism between the CDV-R01 and CDV-H. Our work not only provides a practical toolkit for addressing viral variability but also delivers critical theoretical insights and a rational framework for designing high-potency neutralizing antibodies against CDV.

4. Materials and Methods

4.1. Cell Lines

The Vero cell line (African green monkey kidney epithelial cells) was obtained from the laboratory of Mingqian Feng (Huazhong Agricultural University, Wuhan, China) and was cultured in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; HyGene, Logan, UT, USA), 1% L-glutamine, and 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The Expi-293F cell line (human embryonic kidney cells) was grown in OPM-293 CD05 medium (OPM, Shanghai, China) and incubated under the same conditions of 37 °C and 5% CO2 with balanced air. To establish a cell model susceptible to canine distemper virus (CDV) infection, a Vero cell line stably overexpressing the full-length dog signaling lymphocytic activation molecule (SLAM) was generated via lentiviral transduction. This resulting cell line, designated Vero-SLAM, was cultured under the same conditions as the parental Vero cells.

4.2. CDV Production

Canine distemper virus (CDV) was propagated in the Vero-SLAM cell line. Briefly, nasal secretions collected from a CDV-infected dog were diluted in phosphate-buffered saline (PBS), filtered through a 0.22 μm membrane to remove bacteria and debris, and then inoculated onto Vero-SLAM monolayers. The infected cells were maintained in serum-free DMEM and incubated at 37 °C with 5% CO2 for one week. Successful viral propagation was confirmed by the appearance of extensive cytopathic effects (CPEs), characterized by cell rounding and syncytia formation. Subsequently, the cell culture supernatants containing viral particles were harvested and concentrated 100-fold by high-speed centrifugation at 50,000× g using an Optima XE-90 ultracentrifuge (Beckman, Brea, CA, USA). The resulting virus pellet was resuspended in a suitable volume of PBS, aliquoted, and stored at −80 °C until use.

4.3. Viral Titration and Neutralization Assay by Plaque Method

The viral titers of wild-type canine distemper virus (CDV) isolates were quantified using a plaque assay. Monolayers of Vero-SLAM cells in 6-well plates were inoculated with 200 μL of ten-fold serial dilutions of virus stocks in serum-free DMEM. Following adsorption at 37 °C for 1–2 h with gentle rocking at 15 min intervals, the inoculum was removed and cells were overlaid with semi-solid maintenance medium containing 1.5% carboxymethyl cellulose. After 5 days of incubation at 37 °C in 5% CO2, the overlay was removed and cells were fixed with 10% formalin followed by staining with 0.1% crystal violet. Plaques were counted and viral titers were expressed as plaque-forming units per milliliter (PFU/mL).
For neutralization assessment, a plaque reduction neutralization test was performed. CDV at approximately 50–100 PFU/well was mixed with equal volumes of serially diluted rabbit anti-CDV polyclonal antibody or control serum and incubated at 37 °C for 1 h. The mixtures were then applied to Vero-SLAM cell monolayers and processed through the standard plaque assay procedure as described above. The neutralization titer (PRNT50) was defined as the highest antibody dilution that resulted in a 50% reduction in plaque counts compared to virus-only controls.

4.4. Expression and Purification of Recombinant SLAM and CDV-R01 Proteins

The extracellular domain of canine signaling lymphocyte activation molecule (SLAM) (NP_001003084.1, amino acids 27–237) was fused with human IgG1-Fc and cloned into the mammalian expression vector pFUSE-hIgG1-Fc2 (InvivoGen, San Diego, CA, USA) to generate SLAM-hFc. Similarly, the coding sequence of the selected single-chain variable fragment (scFv) was fused with human IgG1-Fc and cloned into the same vector to generate scFv-hFc.
Recombinant proteins were expressed in Expi293F cells transfected with the respective plasmids using polyethylenimine (PEI; Polysciences, Inc., cat. no. 23966-1, Warrington, PA, USA). The cell culture supernatant was harvested one week post-transfection and clarified by centrifugation at 10,000× g for 1 h at 4 °C, followed by filtration through 0.2-μm membranes.
Both SLAM-hFc and CDV-R01 (scFv-hFc) were purified using Protein A affinity chromatography (Sangon Biotech Co., Ltd., Shanghai, China). After washing with PBS, bound proteins were eluted with low-pH buffer (0.1 M glycine-HCl, pH 2.7) and immediately neutralized with 1 M Tris-HCl (pH 9.0). Protein concentration was determined by BCA assay, and purity was verified by SDS-PAGE with Coomassie Blue staining.

4.5. Panning of the Phage-Displayed Library for CDV-Binding Antibodies

Two chickens (Animal Facility of Huazhong Agricultural University, Wuhan, China) were immunized with 100 μg recombinant SLAM-hFc protein to produce anti-SLAM antibodies for cell line development. Immunization involved a primary subcutaneous injection emulsified in Complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA; Merck KGaA, Darmstadt, Germany), followed by two boosters of half the antigen dose in Incomplete Freund’s adjuvant at 14-day intervals.
After establishing the Vero-SLAM cell line with these antibodies, wild-type canine distemper virus (CDV) was isolated and amplified. Two New Zealand White rabbits (3–4 months old, 1.5–2.0 kg; Animal Facility of Huazhong Agricultural University) were then immunized with 2 mL purified CDV (titer: 1 × 108 PFU/mL) using the same immunization protocol.
Seven days after the final booster, rabbits were euthanized by ear vein injection of sodium pentobarbital (100 mg/kg). Spleens were harvested, and total RNA was extracted from splenocytes with TRIzol reagent (Invitrogen, Carlsbad, CA, USA; Thermo Fisher Scientific, Inc., Osaka, Japan). Following reverse transcription to cDNA, variable region genes of heavy and light chains (VH and VL) were amplified by PCR and assembled into single-chain variable fragments (scFv) using splicing by overlap extension PCR.
The scFv fragments were cloned into pComb3X phagemid vector via SfiI restriction sites and electroporated into E. coli TG1 cells (Shanghai Weidi Biotechnology Co., Ltd., Shanghai, China), yielding a primary library of 2 × 108 independent transformants. For biopanning against CDV, immunotubes were coated with purified CDV virions. After three rounds of panning with increasing wash stringency using PBS containing 0.1% Tween-20, antigen-specific clones were identified by monoclonal phage ELISA.

4.6. Enzyme-Linked Immunosorbent Assay (ELISA)

A 96-well MaxiSorp plate was coated with 5 μg/mL SLAM-hFc or 10 μg/mL CDV virions and incubated at 37 °C for 30 min. After washing with PBST, the plate was blocked with 2% BSA for 30 min. Serially diluted scFv antibodies were added and incubated for 1 h at 37 °C. Bound antibodies were detected using HRP-conjugated anti-His tag antibody (1:4000; Invitrogen) for His-tagged scFvs or HRP-conjugated protein A (1:4000; Invitrogen) for SLAM-hFc. TMB substrate was used for development, the reaction was stopped with 2 M H2SO4, and absorbance was measured at 450 nm.

4.7. Flow Cytometry Method

Vero and Vero-SLAM cells were harvested and incubated with the selected anti-SLAM polyclonal antibody (10 μg/mL) for 1 h on ice. After washing with PBS, cell-bound antibodies were detected using PE-conjugated goat anti-rabbit polyclonal antibody (1:1000; cat. no. ab72465, Abcam, Cambridge, UK). The fluorescence intensity was measured on a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). SLAM expression levels in the engineered cell line were quantified based on PE fluorescence intensity. Unstained cells and cells incubated with secondary antibody alone served as negative controls.

4.8. Molecular Docking and Dynamics Simulations

The three-dimensional structure of the scFv domain of the CDV-R01 fusion protein (excluding the Fc region) was predicted using AlphaFold3 (https://alphafoldserver.com, accessed on 10 September 2025) [36]. For the CDV-H, a homology model was generated based on the sequenced viral strain using MODELLER (https://salilab.org/modeller/download_installation.htm, accessed on 13 October 2025) software, with the electron microscopy structure of CDV-H (PDB entry: 7ZNY) as a template [22,37]. Molecular docking was performed with HADDOCK 2.4 (https://wenmr.science.uu.nl/haddock2.4, accessed on 10 January 2025), defining the CDR-H3 and CDR-L3 loops of the scFv as active residues and specifying key CDV-H residues involved in SLAM receptor binding based on previous studies as the binding interface [38,39].
The top-ranked complex structure from the highest-scoring cluster was selected for molecular dynamics simulations. Simulations were conducted using GROMACS 2024.06 (https://www.gromacs.org, accessed on 10 September 2025) with the AMBER 99 SB-ILDN force field [40]. The complex was solvated in a cubic box of 1.2 nm with 150 mM NaCl. Energy minimization was performed using the steepest descent algorithm until the maximum force reached <1000 kJ/mol/nm. Systems were equilibrated with 100 ps of NVT and NPT ensembles (310 K, 1 bar) using the leap-frog integrator, followed by a 100 ns production MD simulation.
Trajectory analysis included root-mean-square deviation (RMSD), radius of gyration (Rg), solvent-accessible surface area (SASA), hydrogen bonding (Hbond), and free energy landscape (FEL) to evaluate complex stability. The binding free energy and key interacting residues were calculated using gmx_MMPBSA v1.6.3 (https://valdes-tresanco-ms.github.io/gmx_MMPBSA/dev/installation, accessed on 13 October 2025) [41].

4.9. RNA Extraction and Quantitative RT-PCR

RNA was extracted from parental Vero and Vero-SLAM cells using TRIzol reagent (Cat. 15596018, Invitrogen) according to the manufacturer’s instructions. For reverse transcription, 500 ng of total RNA was used (Cat. 28025021, Thermo Fisher). Quantitative RT-PCR was performed using a qRT-PCR kit (RR820L, Takara, Osaka, Japan) on a Bio-Rad CFX96 Real-Time PCR Detection System (Hercules, CA, USA). The reaction conditions were: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The specific primers used are listed below:
Canine SLAM (codon-optimized):
Forward: 5′-GAGCCTGACCATCTATGCCC-3′
Reverse: 5′-CTCTGTTGCTGCCACGTAGA-3′
GAPDH (Internal Control):
Forward: 5′-GTCTCCTCTGACTTCAACAGCG-3′
Reverse: 5′-ACCACCCTGTTGCTGTAGCCAA-3′

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27041895/s1.

Author Contributions

Author Contributions: Conceptualization, M.F.; methodology, M.F., X.C. and J.X.; software, J.X.; validation, J.X., X.H., S.C., Z.Z. (Zhenhuan Zhao), Z.W., M.W. and Z.T.; formal analysis, J.X.; investigation, J.X.; resources, M.F., X.C. and Z.Z. (Zhiyuan Zhao); data curation, J.X.; writing—original draft preparation, J.X.; writing—review and editing, J.X., X.C., M.F. and Z.Z. (Zhiyuan Zhao); visualization, J.X.; supervision, X.C. and Z.Z. (Zhiyuan Zhao); funding acquisition, X.C. and Z.Z. (Zhiyuan Zhao). All authors have read and agreed to the published version of the manuscript.

Funding

The authors declare financial support was received for the research and/or publication of this article. This work was funded by the National Natural Science Foundation of China (32300783), the Hubei Provincial Department of Education Science and Technology Research Project Q20241402, the Open Project Funding of the Key Laboratory of Fermentation Engineering, Ministry of Education (202509FE07, 202509FE08).

Institutional Review Board Statement

All the procedures used in the animal studies were approved by the Animal Care and Use Committee (ACUC) of Huazhong Agricultural University (HZAUMO-2023-0304, 3 April 2023).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Acknowledgments

Supported (or partially supported) by Open Project Funding of the Key Laboratory of Fermentation Engineering (Ministry of Education).

Conflicts of Interest

The authors declare no competing interests.

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Figure 1. Generation and validation of canine SLAM-expressing Vero cells. (A) SDS-PAGE analysis of recombinant canine SLAM extracellular domain (ECD)-hFc protein expressed in Expi-293F cells under non-reducing (Non-re) and reducing (Re) conditions. Molecular weight markers (kDa) are indicated on the left. (B) Serum titer of chicken C037 immunized with the recombinant SLAM (ECD)-hFc. The control antigen, IAB-hFc, was a recombinant fragment of mesothelin. M0, pre-immunization serum; M2, post-immunization serum. Binding is shown for M2 to SLAM (black circles) or IAB (open circles), and M0 to SLAM (black triangles) or IAB (open triangles). Data are presented as mean ± SD of three independent biological replicates. (C) Flow cytometry analysis of surface SLAM expression in sorted Vero-SLAM monoclonal clones. Histograms represent PE-conjugated anti-SLAM staining in unstained Vero cells (black dashed line), unstained Vero-SLAM cells (green), mock-stained parental Vero cells (blue), and SLAM-stained sorted clones (red) cells for specimens 001, 002, 003, and 004; geometric mean fluorescence intensities (Geo Mean PE-A) are provided in the table. (D) Relative mRNA expression of SLAM in parental Vero cells (black bar; negligible expression) versus puromycin-selected Vero-SLAM stable transfectants (gray bar), as determined by qRT-PCR (n = 3; mean ± SD).
Figure 1. Generation and validation of canine SLAM-expressing Vero cells. (A) SDS-PAGE analysis of recombinant canine SLAM extracellular domain (ECD)-hFc protein expressed in Expi-293F cells under non-reducing (Non-re) and reducing (Re) conditions. Molecular weight markers (kDa) are indicated on the left. (B) Serum titer of chicken C037 immunized with the recombinant SLAM (ECD)-hFc. The control antigen, IAB-hFc, was a recombinant fragment of mesothelin. M0, pre-immunization serum; M2, post-immunization serum. Binding is shown for M2 to SLAM (black circles) or IAB (open circles), and M0 to SLAM (black triangles) or IAB (open triangles). Data are presented as mean ± SD of three independent biological replicates. (C) Flow cytometry analysis of surface SLAM expression in sorted Vero-SLAM monoclonal clones. Histograms represent PE-conjugated anti-SLAM staining in unstained Vero cells (black dashed line), unstained Vero-SLAM cells (green), mock-stained parental Vero cells (blue), and SLAM-stained sorted clones (red) cells for specimens 001, 002, 003, and 004; geometric mean fluorescence intensities (Geo Mean PE-A) are provided in the table. (D) Relative mRNA expression of SLAM in parental Vero cells (black bar; negligible expression) versus puromycin-selected Vero-SLAM stable transfectants (gray bar), as determined by qRT-PCR (n = 3; mean ± SD).
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Figure 2. Construction of an anti-CDV scFv phage display library. (A,B) ELISA titration curves of antigen-binding titers in serial dilutions of pre-immune and post-immune sera from rabbits R064 (A) and R065 (B) against CDV-Shamo antigen. Error bars represent the mean ± SD derived from three independent biological replicates. (C) Plaque reduction neutralization assay on Vero-SLAM cells infected with each CDV strain (rows: Jinmao, Shamo, Hashiqi) in the presence of naive rabbit serum (left panels), R064 serum (middle), or R065 serum (right); plaques were visualized by crystal violet staining. R064 serum showed the strongest neutralization across strains. (D) Schematic of scFv library construction from R064 spleen RNA: Blue arrows indicate the experimental workflow. PCR-amplified VL (blue) and VH (red) genes were linked (yellow) to generate scFv, cloned into pComb3X, and electroporated into E. coli TG1. Agarose gels (lower panels) verify VL (~350 bp), VH (~400 bp), and scFv-pComb3X plasmid (~3500 bp) constructs; molecular weight markers (bp) are indicated.
Figure 2. Construction of an anti-CDV scFv phage display library. (A,B) ELISA titration curves of antigen-binding titers in serial dilutions of pre-immune and post-immune sera from rabbits R064 (A) and R065 (B) against CDV-Shamo antigen. Error bars represent the mean ± SD derived from three independent biological replicates. (C) Plaque reduction neutralization assay on Vero-SLAM cells infected with each CDV strain (rows: Jinmao, Shamo, Hashiqi) in the presence of naive rabbit serum (left panels), R064 serum (middle), or R065 serum (right); plaques were visualized by crystal violet staining. R064 serum showed the strongest neutralization across strains. (D) Schematic of scFv library construction from R064 spleen RNA: Blue arrows indicate the experimental workflow. PCR-amplified VL (blue) and VH (red) genes were linked (yellow) to generate scFv, cloned into pComb3X, and electroporated into E. coli TG1. Agarose gels (lower panels) verify VL (~350 bp), VH (~400 bp), and scFv-pComb3X plasmid (~3500 bp) constructs; molecular weight markers (bp) are indicated.
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Figure 3. Expression and characterization of CDV-R01, an anti-CDV scFv-hFc fusion protein. CDV-R01 denotes the fusion construct comprising the screened anti-CDV scFv linked to the human Fc domain (hFc) for enhanced detection and purification. (A) The protein was analyzed under non-reducing and reducing conditions to verify its oligomeric state. Under non-reducing conditions (Lane 1), a predominant band is observed at approximately 110 kDa, corresponding to the disulfide-linked homodimer. Under reducing conditions (Lane 2), the protein migrates as a monomer at approximately 55 kDa. M: Molecular weight marker (kDa). (B) Dose-dependent ELISA binding of serial dilutions of CDV-R01 to immobilized antigens from the three isolated CDV strains (Shamo, red; Jinmao, green; Hashiqi, purple) and vaccine strain (black). Results are expressed as mean ± SD (n = 3). (C) IHC analysis of lung sections from non-infected control dogs (left panels, no specific staining) and CDV-infected dogs (right panels, brown positive staining) using CDV-R01 (400× magnification; scale bar, 50 μm). (D) Western blot detection of CDV antigens in cell lysates from the three isolated strains (Jinmao, Shamo, Hashiqi) and vaccine strain using CDV-R01 (top panel, ~70 kDa); (E) Plaque reduction neutralization assay demonstrating the inhibitory effect of scFv CDV-R01 on CDV infection. Vero-SLAM cells were infected with the CDV-Hashiqi strain in the absence (control, left) or presence (right) of scFv CDV-R01. Following incubation, cells were overlaid with agarose and stained with crystal violet. Representative images show abundant plaque formation (visible as white spots) and cytopathic effects in the control group, whereas plaque formation was markedly reduced in the CDV-R01-treated group, indicating that CDV-R01 effectively inhibits CDV infection of Vero-SLAM cells.
Figure 3. Expression and characterization of CDV-R01, an anti-CDV scFv-hFc fusion protein. CDV-R01 denotes the fusion construct comprising the screened anti-CDV scFv linked to the human Fc domain (hFc) for enhanced detection and purification. (A) The protein was analyzed under non-reducing and reducing conditions to verify its oligomeric state. Under non-reducing conditions (Lane 1), a predominant band is observed at approximately 110 kDa, corresponding to the disulfide-linked homodimer. Under reducing conditions (Lane 2), the protein migrates as a monomer at approximately 55 kDa. M: Molecular weight marker (kDa). (B) Dose-dependent ELISA binding of serial dilutions of CDV-R01 to immobilized antigens from the three isolated CDV strains (Shamo, red; Jinmao, green; Hashiqi, purple) and vaccine strain (black). Results are expressed as mean ± SD (n = 3). (C) IHC analysis of lung sections from non-infected control dogs (left panels, no specific staining) and CDV-infected dogs (right panels, brown positive staining) using CDV-R01 (400× magnification; scale bar, 50 μm). (D) Western blot detection of CDV antigens in cell lysates from the three isolated strains (Jinmao, Shamo, Hashiqi) and vaccine strain using CDV-R01 (top panel, ~70 kDa); (E) Plaque reduction neutralization assay demonstrating the inhibitory effect of scFv CDV-R01 on CDV infection. Vero-SLAM cells were infected with the CDV-Hashiqi strain in the absence (control, left) or presence (right) of scFv CDV-R01. Following incubation, cells were overlaid with agarose and stained with crystal violet. Representative images show abundant plaque formation (visible as white spots) and cytopathic effects in the control group, whereas plaque formation was markedly reduced in the CDV-R01-treated group, indicating that CDV-R01 effectively inhibits CDV infection of Vero-SLAM cells.
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Figure 4. Structural modeling of the CDV-R01 (derived from the CDV-R01 scFv-hFc fusion protein) and docking with CDV-H. (A) AlphaFold3-predicted structure of the CDV-R01 variable domain, colored by pLDDT confidence score (darker blue indicates higher confidence). CDR-H3 and CDR-L3 loops are highlighted as hypervariable regions implicated in antigen recognition. (B) Ramachandran plot evaluating stereochemical quality of the scFv model. The plot regions are color-coded as follows: red for the most favored regions, yellow for additional allowed regions, pale yellow for generously allowed regions, and white for disallowed regions. Residues are represented by symbols: black squares indicate non-glycine and non-proline residues, while black triangles indicate glycine residues. Of 186 non-Gly and non-Pro residues, 169 (90.9%) reside in most favored regions, 15 (8.1%) in additional allowed regions, 0 (0.0%) in generously allowed regions, and 2 (1.1%) in disallowed regions (likely terminal residues), confirming excellent overall backbone geometry with all residues in acceptable conformational space. (C) Representative structure from the top-scoring HADDOCK cluster (cluster 2 of 7) among 170 docked CDV-H/CDV-R01 poses. The complex is shown in surface (top left) and cartoon (bottom right) representations, with the CDV-H in cyan and the CDV-R01 in green; key interacting residues at the interface are labeled (right panel): CDV-R01 CDR-H3 residues V96, Y98 and N99 interact with CDV-H residues R529, S546 and S194, respectively, while CDV-R01 CDR-L3 residue S224 interacts with CDV-H residue G190.
Figure 4. Structural modeling of the CDV-R01 (derived from the CDV-R01 scFv-hFc fusion protein) and docking with CDV-H. (A) AlphaFold3-predicted structure of the CDV-R01 variable domain, colored by pLDDT confidence score (darker blue indicates higher confidence). CDR-H3 and CDR-L3 loops are highlighted as hypervariable regions implicated in antigen recognition. (B) Ramachandran plot evaluating stereochemical quality of the scFv model. The plot regions are color-coded as follows: red for the most favored regions, yellow for additional allowed regions, pale yellow for generously allowed regions, and white for disallowed regions. Residues are represented by symbols: black squares indicate non-glycine and non-proline residues, while black triangles indicate glycine residues. Of 186 non-Gly and non-Pro residues, 169 (90.9%) reside in most favored regions, 15 (8.1%) in additional allowed regions, 0 (0.0%) in generously allowed regions, and 2 (1.1%) in disallowed regions (likely terminal residues), confirming excellent overall backbone geometry with all residues in acceptable conformational space. (C) Representative structure from the top-scoring HADDOCK cluster (cluster 2 of 7) among 170 docked CDV-H/CDV-R01 poses. The complex is shown in surface (top left) and cartoon (bottom right) representations, with the CDV-H in cyan and the CDV-R01 in green; key interacting residues at the interface are labeled (right panel): CDV-R01 CDR-H3 residues V96, Y98 and N99 interact with CDV-H residues R529, S546 and S194, respectively, while CDV-R01 CDR-L3 residue S224 interacts with CDV-H residue G190.
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Figure 5. Molecular dynamics simulation of the CDV-H/CDV-R01 complex over 100 ns. (A) Root-mean-square deviation (RMSD) trajectories for the CDV-H/CDV-R01 complex (red), isolated CDV-H (blue), and isolated CDV-R01 (green), indicating overall structural stability. (B) Time-dependent number of hydrogen bonds (red, cutoff ≤ 0.35 nm) and close residue pairs (blue, cutoff ≤ 0.35 nm) between CDV-H and CDV-R01, highlighting persistent interactions. (C) Free energy landscape (FEL) projected onto RMSD and radius of gyration (Rg) coordinates, with binding free energy (Gibbs free energy) (kcal/mol) identifying the global minimum; superposition of the representative low-energy structure (cyan: CDV-H; green: scFv) onto the initial docking model (gray). (D) The minimum-energy structure reveals hydrogen bonds (yellow dashes) and a salt bridge (red dashes) between CDV-H (cyan carbon atoms) and scFv (green carbon atoms) residues. Specific interactions include hydrogen bonds between scFv CDR-H3 residues V96, Y98, and N99 and CDV-H residues T548, T192, and S194, respectively. Additionally, a critical salt bridge forms between scFv CDR-H3 residue D103 and CDV-H residue R529, while scFv CDR-L3 residue S224 forms a hydrogen bond with CDV-H residue T192. (E) Per-residue free energy decomposition (ΔG, kcal/mol) for CDV-H (R: receptor) and CDV-R01 (L: ligand) contributions to binding stability during the 100 ns simulation. Notably, residue Y98 in the scFv CDR-H3 loop exhibits the highest contribution to the total binding energy.
Figure 5. Molecular dynamics simulation of the CDV-H/CDV-R01 complex over 100 ns. (A) Root-mean-square deviation (RMSD) trajectories for the CDV-H/CDV-R01 complex (red), isolated CDV-H (blue), and isolated CDV-R01 (green), indicating overall structural stability. (B) Time-dependent number of hydrogen bonds (red, cutoff ≤ 0.35 nm) and close residue pairs (blue, cutoff ≤ 0.35 nm) between CDV-H and CDV-R01, highlighting persistent interactions. (C) Free energy landscape (FEL) projected onto RMSD and radius of gyration (Rg) coordinates, with binding free energy (Gibbs free energy) (kcal/mol) identifying the global minimum; superposition of the representative low-energy structure (cyan: CDV-H; green: scFv) onto the initial docking model (gray). (D) The minimum-energy structure reveals hydrogen bonds (yellow dashes) and a salt bridge (red dashes) between CDV-H (cyan carbon atoms) and scFv (green carbon atoms) residues. Specific interactions include hydrogen bonds between scFv CDR-H3 residues V96, Y98, and N99 and CDV-H residues T548, T192, and S194, respectively. Additionally, a critical salt bridge forms between scFv CDR-H3 residue D103 and CDV-H residue R529, while scFv CDR-L3 residue S224 forms a hydrogen bond with CDV-H residue T192. (E) Per-residue free energy decomposition (ΔG, kcal/mol) for CDV-H (R: receptor) and CDV-R01 (L: ligand) contributions to binding stability during the 100 ns simulation. Notably, residue Y98 in the scFv CDR-H3 loop exhibits the highest contribution to the total binding energy.
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Table 1. Statistical analysis of the lowest energy clusters from HADDOCK docking of the CDV-R01/CDV-H complexes.
Table 1. Statistical analysis of the lowest energy clusters from HADDOCK docking of the CDV-R01/CDV-H complexes.
ClusterSize 1RMSD 2 (Å)Intermolecular Energy, kcal/mol
Average/SD		Buried Surface Area 4, Å 2HADDOCK Score 4
Vdw 3Elec 3AIR 3
1540.6/0.4−81.6/5.9−234.5/11.0172.7/38.72617.6/90.5−115.0/9.7
24617.4/0.1−88.4/4.7−262.6/46.0142.7/47.52650.9/62.6−123.1/2.5
3434.5/0.2−62.6/4.6−190.6/23.2168.6/10.22154.1/61.7−90.8/2.8
4133.3/0.5−44.7/5.7−215.5/10.8142.0/30.11900.2/217.9−75.7/5.8
5615.2/0.6−51.8/7.2−190.1/20.0204.7/31.51996.9/71.9−71.6/8.6
643.1/0.4−39.9/11.6−121.8/28.6166.9/28.31827.7/300.4−41.8/18.6
748.9/0.8−59.2/6.1−241.0/14.3184.2/52.32260.3/57.6−85.0/5.1
Note: 1 Number of structural models contained within each cluster. 2 Values represent the mean ± standard deviation, calculated relative to the lowest-energy structure identified within the same cluster. 3 Intermolecular interaction energies—Van der Waals (Vdw), electrostatic (Elec), and AIR—computed by HADDOCK for each cluster. 4 Clusters are numbered following HADDOCK’s default procedure for automatic enumeration of representative structures.
Table 2. MM/GBSA calculation of the binding free energy for the CDV-R01-CDV-H interaction.
Table 2. MM/GBSA calculation of the binding free energy for the CDV-R01-CDV-H interaction.
ContributionCDV-R01/CDV-H (kcal/mol)
ΔVDWAALS−126.54
ΔEEL−88.93
ΔEGB97.56
ΔESURF−19.41
ΔGGAS−215.47
ΔGSOLV78.15
ΔGtotal−137.32
Note: The contributions to the binding free energy (ΔGtotal) from van der Waals and electrostatic interactions are represented by ΔEvdw and ΔEele, respectively. The polar solvation energy (Generalized Born component) and nonpolar solvation energy (surface area term) contributions to ΔGtotal are represented by ΔEGB and ΔESURF, respectively. ΔGgas denotes the total gas-phase energy (ΔEvdw + ΔEele), and ΔGsolv denotes the total solvation free energy (ΔEGB + ΔESURF). All values are in kcal/mol.
Table 3. Hydrogen bonds and salt bridges between CDV-R01 and CDV-H in the CDV-R01/CDV-H complex.
Table 3. Hydrogen bonds and salt bridges between CDV-R01 and CDV-H in the CDV-R01/CDV-H complex.
ChainResidueChainResidueInteraction Type
CDV-R01SER72.OGCDV-HGLN495.OHydrogen bond
CDV-R01VAL96.HG1CDV-HTHR548.HG1Hydrogen bond
CDV-R01TYR98.HHCDV-HTHR192.OHydrogen bond
CDV-R01ASN99.OD1CDV-HSER194.HHydrogen bond
CDV-R01ASN99.HD22CDV-HSER194.OGHydrogen bond
CDV-R01ASN99.HD22CDV-HSER194.OHydrogen bond
CDV-R01ASP103.OD1CDV-HARG529.HH21Hydrogen bond
CDV-R01ASP103.OD2 CDV-HARG529.HE Hydrogen bond
CDV-R01ASP103.OD1 CDV-HARG529.HH21Hydrogen bond
CDV-R01ASP103.OD2 CDV-HARG529.HE Hydrogen bond
CDV-R01ASP103.OD1CDV-HARG529.NE Salt bridge
CDV-R01ASP103.OD1CDV-HARG529.NH2Salt bridge
CDV-R01ASP103.OD2CDV-HARG529.NE Salt bridge
CDV-R01ASP103.OD2CDV-HARG529.NH2Salt bridge
CDV-R01SER130.HG CDV-HSER189.HO Hydrogen bond
CDV-R01GLN157.HE22 CDV-HARG604.O Hydrogen bond
CDV-R01GLU185.OE2 CDV-HARG552.HH22Hydrogen bond
CDV-R01GLU185.OE2 CDV-HARG552.HH12Hydrogen bond
CDV-R01GLU185.OE2CDV-HARG552.NH2Salt bridge
CDV-R01GLU185.OE2CDV-HARG552.NH1Salt bridge
CDV-R01SER186.HG CDV-HASP531.OD2Hydrogen bond
CDV-R01SER224.OG CDV-HTHR192.HHydrogen bond
CDV-R01SER72.OGCDV-HGLN495.OHydrogen bond
CDV-R01VAL96.HG1CDV-HTHR548.HG1Hydrogen bond
CDV-R01TYR98.HH CDV-HTHR192.OHydrogen bond
CDV-R01ASN99.OD1 CDV-HSER194.H Hydrogen bond
CDV-R01ASN99.HD22 CDV-HSER194.OGHydrogen bond
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Xu, J.; Hu, X.; Chen, S.; Zhao, Z.; Wang, Z.; Wang, M.; Tang, Z.; Feng, M.; Zhao, Z.; Chen, X. Construction and Application of a Canine SLAM Receptor-Based System from Vero Cell Line to Virus Isolation and Parallel Antibody Screening. Int. J. Mol. Sci. 2026, 27, 1895. https://doi.org/10.3390/ijms27041895

AMA Style

Xu J, Hu X, Chen S, Zhao Z, Wang Z, Wang M, Tang Z, Feng M, Zhao Z, Chen X. Construction and Application of a Canine SLAM Receptor-Based System from Vero Cell Line to Virus Isolation and Parallel Antibody Screening. International Journal of Molecular Sciences. 2026; 27(4):1895. https://doi.org/10.3390/ijms27041895

Chicago/Turabian Style

Xu, Jianbo, Xiangda Hu, Sizhuo Chen, Zhenhuan Zhao, Zhangchang Wang, Minghui Wang, Zhongchuan Tang, Mingqian Feng, Zhiyuan Zhao, and Xin Chen. 2026. "Construction and Application of a Canine SLAM Receptor-Based System from Vero Cell Line to Virus Isolation and Parallel Antibody Screening" International Journal of Molecular Sciences 27, no. 4: 1895. https://doi.org/10.3390/ijms27041895

APA Style

Xu, J., Hu, X., Chen, S., Zhao, Z., Wang, Z., Wang, M., Tang, Z., Feng, M., Zhao, Z., & Chen, X. (2026). Construction and Application of a Canine SLAM Receptor-Based System from Vero Cell Line to Virus Isolation and Parallel Antibody Screening. International Journal of Molecular Sciences, 27(4), 1895. https://doi.org/10.3390/ijms27041895

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