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Emerging microbes & infections2024; 13(1); 2396864; doi: 10.1080/22221751.2024.2396864

A platform for the rapid screening of equine immunoglobins F (ab)2 derived from single equine memory B cells able to cross-neutralize to influenza virus.

Abstract: Single B cells-based antibody platforms offer an effective approach for the discovery of useful antibodies for therapeutic or research purposes. Here we present a method for screening equine immunoglobins F(ab)2, which offers the potential advantage of reacting with multiple epitopes on the virus. Using equine influenza virus (EIV) as model, a hemagglutinin (HA) trimer was constructed to bait B cells in vaccinated horses. We screened 370 HA-specific B cells from 1 × 10 PBMCs and identified a diverse set of equine variable region gene sequences of heavy and light chains and then recombined with humanized Ig Fc. Recombinant equine Ig was then self-assembled in co-transfected 293 T cells, and subsequently optimized to obtain HA binding B-cell receptor (s). The recombinant antibodies exhibited a high binding affinity to the HA protein. Antibody H81 exhibited the highest cross neutralizing activities against EIV strains . Furthermore, it effectively protected EIV-challenged mice, resulting in significantly improved survival, reduced pulmonary inflammation and decreased viral titers. predication identified a functional region of H81 comprising 27 key amino acids cross the main circulating EIV strains. The 12 amino acid residues in this region with the highest binding affinities were screened. Notably, the predicted epitopes of H81 encompassed the documented equine HA receptor binding site, validating its cross-neutralization. In summary, a rapid platform was successfully established to investigate the profiling of equine antigen-recognizing receptors (BCRs) following infection. This platform has the potential to optimize the screening of virus-neutralizing antibodies and aid in vaccine design.
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Publication Date: 2024-09-27 PubMed ID: 39331815PubMed Central: PMC11441081DOI: 10.1080/22221751.2024.2396864Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This research presents a rapid screening platform for identifying equine immunoglobulin F(ab)2 antibodies from single equine memory B cells that can broadly neutralize equine influenza virus (EIV).
  • The platform was used to discover antibodies with cross-protective capabilities, and one such antibody (H81) was shown to effectively protect mice from EIV infection.

Background and Significance

  • Single B cell-based antibody discovery platforms allow the isolation of specific antibodies with therapeutic or research potential.
  • Equine influenza virus (EIV) causes respiratory disease in horses, and broad neutralization antibodies would be valuable for treatment and vaccine development.
  • F(ab)2 fragments, which are antibody parts containing two antigen-binding sites without the Fc region, can bind multiple epitopes, potentially enhancing neutralization.

Methodology

  • Constructed an EIV hemagglutinin (HA) trimer protein used as bait to isolate HA-specific memory B cells from vaccinated horses.
  • Screened 370 HA-specific B cells isolated from peripheral blood mononuclear cells (PBMCs) of vaccinated horses.
  • Sequenced the variable regions of the heavy and light chains from these B cells to identify diverse equine antibody gene sequences.
  • Recombined these variable regions with humanized Ig Fc regions to create recombinant antibodies suitable for expression and functional testing.
  • Co-transfected recombinant antibodies into 293T cells for antibody expression and self-assembly.
  • Optimized the expressed F(ab)2 fragments to enhance the ability to bind HA protein, thus improving their affinity and neutralizing function.

Key Findings

  • The recombinant antibodies demonstrated high affinity binding to the influenza HA protein.
  • Among the antibodies, one named H81 exhibited the strongest cross-neutralizing activity against multiple EIV strains.
  • In vivo experiments with EIV-challenged mice showed that H81 antibody treatment:
    • Improved survival rates significantly.
    • Reduced lung inflammation caused by the virus.
    • Decreased viral load in lung tissues.
  • Epitope mapping and prediction identified a functional region in H81 composed of 27 critical amino acids conserved across major circulating EIV strains.
  • From this region, 12 amino acid residues with the highest binding affinities were further identified.
  • The predicted epitopes of H81 overlapped with the known receptor binding site on equine HA, explaining the broad neutralization ability.

Conclusions and Implications

  • This study successfully established a platform for rapid screening and characterization of equine antigen-specific B cell receptors post-infection or vaccination.
  • The platform enables the discovery of broadly neutralizing antibodies capable of cross-protecting against diverse virus strains.
  • The identified antibody H81 could serve as a model for therapeutic development against equine influenza infections.
  • Detailed epitope mapping of effective antibodies aids rational vaccine design by targeting conserved viral sites involved in receptor binding.
  • Overall, the approach enhances the ability to optimize antibody screening and vaccine strategies against equine influenza and potentially other viral diseases of horses.

Cite This Article

APA
Lin Y, Wang Y, Li H, Liu T, Zhang J, Guo X, Guo W, Wang Y, Liu X, Huang S, Liao H, Wang X. (2024). A platform for the rapid screening of equine immunoglobins F (ab)2 derived from single equine memory B cells able to cross-neutralize to influenza virus. Emerg Microbes Infect, 13(1), 2396864. https://doi.org/10.1080/22221751.2024.2396864

Publication

Emerging microbes & infections
ISSN: 2222-1751
NlmUniqueID: 101594885
Country: United States
Language: English
Volume: 13
Issue: 1
Pages: 2396864
PII: 2396864

Researcher Affiliations

Lin, Yuezhi
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
Wang, Yayu
  • Zhuhai Trinomab Pharmaceutical Co., Ltd, Zhuhai, People's Republic of China.
Li, Hongxin
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
Liu, Tong
  • Zhuhai Trinomab Pharmaceutical Co., Ltd, Zhuhai, People's Republic of China.
Zhang, Jiaqi
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
Guo, Xing
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
Guo, Wei
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
  • Institute of Western Agriculture, the Chinese Academy of Agricultural sciences, Changji, People's Republic of China.
Wang, Yaoxin
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
Liu, Xiangning
  • Clinical Research Platform for Interdiscipline of Stomatology, The First Affiliated Hospital of Jinan University, Guangzhou, People's Republic of China.
  • Department of Stomatology, College of Stomatology, Jinan University, Guangzhou, People's Republic of China.
Huang, Shaoli
  • The Hong Kong University of Science and Technology, School of Engineering, Hong Kong, People's Republic of China.
Liao, Huaxin
  • Zhuhai Trinomab Pharmaceutical Co., Ltd, Zhuhai, People's Republic of China.
Wang, XiaoJun
  • State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
  • Institute of Western Agriculture, the Chinese Academy of Agricultural sciences, Changji, People's Republic of China.

MeSH Terms

  • Animals
  • Horses
  • Orthomyxoviridae Infections / veterinary
  • Orthomyxoviridae Infections / prevention & control
  • Orthomyxoviridae Infections / immunology
  • Orthomyxoviridae Infections / virology
  • Mice
  • Antibodies, Viral / immunology
  • B-Lymphocytes / immunology
  • Antibodies, Neutralizing / immunology
  • Humans
  • Hemagglutinin Glycoproteins, Influenza Virus / immunology
  • Hemagglutinin Glycoproteins, Influenza Virus / genetics
  • Influenza A Virus, H3N8 Subtype / immunology
  • Influenza A Virus, H3N8 Subtype / genetics
  • Immunoglobulin Fab Fragments / immunology
  • Immunoglobulin Fab Fragments / genetics
  • Cross Reactions
  • HEK293 Cells
  • Influenza Vaccines / immunology
  • Immunologic Memory
  • Female
  • Epitopes / immunology

Conflict of Interest Statement

No potential conflict of interest was reported by the author(s).

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Citations

This article has been cited 3 times.
  1. Kökkaya S, Göksu AG, Sözdutmaz İ, Toy MA, Berber E. Monoclonal antibodies in veterinary antiviral immunotherapy: technologies, applications and challenges. Vet Res Commun 2025 Oct 22;49(6):366.
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  2. Yu L, Li F, Zou X, Xu L, Zhao J, Li Y, Peng G, Xia Y, Zhao Q, Zhu Y. Development and Application of Mouse-Derived CD2v Monoclonal Antibodies Against African Swine Fever Virus from Single B Cells. Viruses 2025 Aug 15;17(8).
    doi: 10.3390/v17081123pubmed: 40872837google scholar: lookup
  3. Firdausy LW, Fikri F, Wicaksono AP, Çalışkan H, Purnama MTE. Global prevalence and risk factors of equine infectious anemia: A systematic review and meta-analysis. Vet World 2025 Jun;18(6):1440-1451.
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