Aug 2026· Veterinary Sciences· Vol 13, pp. 835· 0 citations· 38 references
Medicine
TL;DR
This study developed two mAbs against porcine CD163 that can inhibit PRRSV infection, while mAb 11D cannot, and provides a solid foundation for elucidating CD163-domain-dependent biological functions.
Abstract
Simple Summary Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes substantial economic losses to the global swine industry. PRRSV invades host target cells via receptor-mediated endocytosis, and CD163 is the most important receptor for PRRSV entry. Among the CD163 domains, the scavenger receptor cysteine-rich 5 (SRCR5) domain is essential for PRRSV infection. Therefore, CD163 and its SRCR5 are commonly selected as targets for developing anti-PRRSV strategies, including for the generation of CD163-SRCR5 knockout pigs. In addition, CD163 divergence also serves as a barrier to cross-species cellular infection by arteriviruses. Nevertheless, CD163 monoclonal antibodies (mAbs) with defined cross-species reactivity and anti-PRRSV activities have rarely been characterized. Here, we developed two mAbs against porcine CD163. Epitope mapping demonstrated that mAb 5A recognizes a novel epitope within the long loop 5-6 of the SRCR5 domain, while mAb 11D targets a conserved epitope within the SRCR4 domain. Functional validation revealed that mAb 5A can inhibit PRRSV infection, while mAb 11D cannot. Remarkably, mAb 11D exhibits broader cross-species reactivity than mAb 5A. Overall, this study provides a solid foundation for elucidating CD163-domain-dependent biological functions.
BACKGROUND
Brucellosis, a severe zoonotic infectious disease, poses substantial economic and health threats globally. The intracellular survival strategy of Brucella complicates disease control, highlighting the need for novel immunotherapeutic strategies such as antibody-based therapies and multi-epitope vaccines.
RESULTS
This study generated two IgM monoclonal antibodies (D3 and F5) against the conserved outer membrane protein OMP16 of Brucella using hybridoma technology. Peptide scanning and Western blot identified their linear epitopes (D3: 77TLSKQAQW84; F5: 120RDFLASRG127), which are highly conserved among major Brucella species. Integrated approaches-including molecular docking, alanine-scanning mutagenesis, and dot-blot assays-revealed key residues at the epitope interface that form stable bonds with antibody complementarity-determining regions (CDRs). Functionally, both antibodies activated the complement system, with F5 exhibiting significant complement-dependent bacteriolytic activity in vitro. Furthermore, in the presence of complement, D3 and F5 enhanced macrophage-mediated opsonophagocytosis and intracellular killing of Brucella abortus A19. In a mouse infection model, passive immunization with either antibody significantly alleviated infection-induced weight loss and splenomegaly and reduced bacterial load in the spleen.
CONCLUSIONS
Our study underscores the role of IgM antibodies in combating Brucella infection, offers insights for antibody-based immunotherapy, and provides a theoretical foundation for developing multi-epitope vaccines based on the conserved epitopes and critical residues.
Yunyi Zhai, Kaihui Sun, Ye Yuan et al.· BMC Biology· 0 citations
H9N2 avian influenza virus (AIV) remains a global threat to poultry health and has zoonotic potential. Antigenic drift in the hemagglutinin (HA) protein complicates vaccine efficacy and diagnostic accuracy, highlighting the need for precise epitope characterization. In this study, the HA protein of H9N2 AIV was expressed in a eukaryotic system, and two monoclonal antibodies (mAbs), 9C12 and 9F4, were generated. Both mAbs specifically bound HA, as shown by ELISA, Western blot, and immunofluorescence, but lacked hemagglutination inhibition activity. Epitope mapping revealed two minimal linear epitopes: 123FSSSRSYQ130 within the vestigial esterase domain and 201NLYTRTDTT209 within the receptor-binding domain. Alanine scanning revealed key residues required for antibody binding, whereas structural modeling confirmed that both epitopes are surface exposed. Sequence analysis demonstrated strong conservation across H9N2 strains, with the 9F4 epitope showing near-complete invariance, whereas both epitopes exhibited low conservation among other influenza A virus subtypes. These findings define two novel, nonneutralizing epitopes on H9N2 HA that expand the antigenic map and represent promising targets for subtype-specific diagnostic assays.
Coronaviruses (CoVs) cause severe respiratory diseases and continue to pose a significant worldwide health threat. Antibodies that specifically target the structural proteins of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) are crucial for immunological research, development of diagnostic assays, and evaluation of immune responses. Avian egg yolk immunoglobulin Y (IgY) represents a novel approach that has attracted considerable interest due to its strong immunological response, scalability, and excellent safety profile. The study involved generating SARS-CoV-2 receptor-binding domain (RBD)-specific spike (S) IgY antibodies by immunising laying hens with a synthetic RBD epitope. IgY antibodies were extracted from egg yolks using optimised purification methods and subsequently characterised for yield, purity, and antigen specificity. The purified IgY preparations were evaluated and verified for specificity and binding affinity to the target antigen using indirect enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), conventional Western blotting, and automated Jess Western blot analysis. The produced antibodies exhibited targeted recognition of the antigen and were effectively purified with commendable yield and purity, highlighting their potential as a cost-effective, scalable, and dependable source of primary antibodies for immunological research and diagnostic applications. Additional research, including virus-neutralisation and in vivo efficacy assessments, is needed to explore their potential for passive immunisation and various biomedical applications.
Dhruvi Patel, K. Babu, Ronak Solanki et al.· Immunobiology· 0 citations
Background Myxovirus resistance (Mx) is a potent antiviral effector induced by interferons, playing a key role in restricting diverse viral infections. However, the specific functions of duck Mx remain poorly understood due to the lack of specific detection reagents. Methods In this study, we cloned the Mx gene from ShaoXing duck and expressed and purified its truncated fragment (Mx-a) in prokaryotic cells. Using this purified recombinant Mx-a protein as an immunogen, we generated a monoclonal antibody (4B6) that specifically recognizes native duck Mx. Results The antibody, an IgG1 subtype, exhibited high titer and excellent specificity. Further investigation confirmed that mAb 4B6 specifically detects endogenous Mx in duck embryonic tissues by Western blot and recognizes Mx expressed in eukaryotic cells by immunofluorescence. Through fine epitope mapping, the linear epitope recognized by mAb 4B6 was precisely defined as the amino acid sequence “IYFPVPEQ” (residues 67–74), which is highly conserved across different duck species. Conclusion The specific monoclonal antibody 4B6 provides an essential tool for further studies on the biological functions, antiviral mechanisms, and innate immune role of duck Mx.
Xinru Song, Shanshan Fan, Zihan Zhu et al.· Frontiers in Veterinary Scie...· 0 citations
Porcine epidemic diarrhea virus (PEDV) causes devastating enteric disease in piglets, yet the mechanistic basis of antibody-mediated neutralization remains poorly understood. Here, we determined the structure of PEDV HNXX-strain spike domain B (S1B) simultaneously bound by C62, a neutralizing porcine monoclonal antibody against PEDV G2 strains, and N34, a non-neutralizing porcine PEDV antibody. The structure reveals that C62 targets a conserved, cryptic epitope that is accessible only when S1B adopts an “up” conformation. Functionally, we showed that C62 has substantially stronger activity than N34 in triggering S-trimer disassembly and inducing the formation of proteinase K-resistant, post-fusion-like S2 structures. Despite the weaker triggering activity of N34, both C62 and N34 can function as artificial receptors. Notably, although the C62 epitope is conserved across both G1 and G2 strains, C62 exhibits G2-strain-biased neutralizing activity. We further showed that differences in cell-surface membrane fusion activity among PEDV spikes correlate with distinct viral entry pathways and are jointly determined by the S1A and S1B sequences. Together, our findings identify a strain-specific vulnerable site on the PEDV S-trimer and provide insight into how cell-surface membrane fusion activity may influence viral entry pathway selection and antibody neutralization efficacy.
Jian-Bo Liu, Sheng Wang, Zimu Li et al.· bioRxiv· 0 citations
ABSTRACT Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that causes severe diarrhea, vomiting, and dehydration in piglets, and a recent human infection report raises concerns about its zoonotic potential. The S1 subunit of the PDCoV-spike protein is the primary target of neutralizing antibodies and is critical for viral attachment and entry. In this study, we generated and characterized eight monoclonal antibodies (mAbs) against S1, two targeting the S1A domain, five targeting the S1B domain, and one binding the S1C domain. Using a genetically engineered luciferase reporter virus, we demonstrated that all five S1B-targeting mAbs neutralize viral infection by disrupting S1-aminopeptidase N receptor engagement. These S1B mAbs cross-react with S1B proteins from multiple human- and avian-origin deltacoronaviruses, suggesting the recognition of conserved antigenic regions. Importantly, we found that our PDCoV strain caused embryo lethality in embryonated chicken eggs (ECEs). Using this ECE lethal model, we performed a preliminary in vivo evaluation of the five S1B-specific neutralizing mAbs. All five mAbs improved embryo survival, with 3E9 providing complete protection against PDCoV-induced embryonic lethality. Collectively, these novel mAbs represent valuable tools for diagnosis and therapeutic development. Moreover, the luciferase-reporter virus and the ECE model establish robust platforms for future evaluation of neutralizing antibodies against PDCoV. IMPORTANCE Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that threatens swine health and poses a potential risk of cross-species transmission; however, effective countermeasures remain limited. We generated and characterized a panel of monoclonal antibodies targeting the PDCoV S1 protein and identified five S1B-specific neutralizing antibodies that cross-react with S1B proteins from multiple human- and avian-origin deltacoronavirusess. We also established complementary in vitro and preliminary in vivo platforms for antibody evaluation using a firefly luciferase reporter virus and an embryonated chicken egg model. Together, these findings expand the repertoire of PDCoV-neutralizing antibodies and provide practical tools for antibody characterization and preliminary protective efficacy assessment, facilitating future studies on antibody-based interventions against emerging deltacoronaviruses. Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that threatens swine health and poses a potential risk of cross-species transmission; however, effective countermeasures remain limited. We generated and characterized a panel of monoclonal antibodies targeting the PDCoV S1 protein and identified five S1B-specific neutralizing antibodies that cross-react with S1B proteins from multiple human- and avian-origin deltacoronavirusess. We also established complementary in vitro and preliminary in vivo platforms for antibody evaluation using a firefly luciferase reporter virus and an embryonated chicken egg model. Together, these findings expand the repertoire of PDCoV-neutralizing antibodies and provide practical tools for antibody characterization and preliminary protective efficacy assessment, facilitating future studies on antibody-based interventions against emerging deltacoronaviruses.
Jiaru Zhou, Ran Jing, Mengdi Zhang et al.· mBio· 0 citations
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