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. 2025 Jun 20;12(7):601.
doi: 10.3390/vetsci12070601.

Preparation of Monoclonal Antibodies Against the gD Protein of Feline Herpesvirus Type-1 by mRNA Immunization

Chengqi Zhang  1 , Yawen Liu  1 , Guangrong Zhao  1 , Bo Hu  1 , Liwen Xu  1 , Jiajia Liu  1 , Yajie Sun  1 , Xiaolan Guo  1 , Xiaoyu Deng  1 , Shizhen Lian  1 , Tiyun Han  2 , Mengwei Xu  2 , Shi Xu  2 , Xue Bai  1
Affiliations

Preparation of Monoclonal Antibodies Against the gD Protein of Feline Herpesvirus Type-1 by mRNA Immunization

Chengqi Zhang et al. Vet Sci. .

Abstract

This study aimed to develop monoclonal antibodies (mAbs) against the gD protein of FHV-1 for rapid and specific virus detection. The gD protein, a highly conserved part of the FHV-1 envelope, is crucial for viral entry into host cells, making it an ideal detection target. We immunized BALB/c mice with an mRNA vaccine encoding the gD gene, achieving a serum antibody titer of 1:140,000 after three immunizations. The mice were then boosted with recombinant gD protein. Through cell fusion and multiple subcloning rounds, we obtained five hybridoma cell lines (D7, E4, E9, E10, and E19) that stably secrete anti-gD protein mAbs. Characterization by indirect immunofluorescence and Western blot showed that mAbs D7 and E4 have high specificity and strong binding activity against FHV-1, detectable at 2 μg/mL. These mAbs provide specific tools for FHV-1 detection and a basis for developing rapid diagnostic methods using ELISA, colloidal gold, and other technologies.

Keywords: clinical diagnosis; feline herpesvirus type-1; gD protein; mRNA vaccine; monoclonal antibody.

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Conflict of interest statement

Authors Tiyun Han, Mengwei Xu and Shi Xu were employed by the company Nanjing Chengshi BioTech (TheraRNA) Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Design, preparation, and validation of protein expression of mRNA vaccine. (A) mRNA vaccine design schematic. (B) In vitro transcription mRNA quality and length determination. (C) Detection of the particle size and polydispersity index of mRNA–LNPs. (D) Zeta potential detection of mRNA–LNP vaccine. (E) Detection of gD protein expression in HEK293T cells transfected with mRNA–LNPs for 24 h. M represents the protein marker. Lane 1 contains untransfected HEK293T cells, and Lane 2 contains HEK293T cells transfected with mRNA–LNPs (original Figure see Supplementary Material).
Figure 2
Figure 2
Evaluation of mRNA vaccine immunogenicity and antigen expression for monoclonal antibody screening. (A) Flowchart of mouse immunization procedure. (B) Expression of gD protein for monoclonal antibody screening. Lane 1: Supernatant from CHO cells transfected with pcDNA3.4-gD. Lane 2: Supernatant from CHO cells transfected with empty pcDNA3.4 vector. (C) Detection of FHV-specific antibody titers in mouse serum. (D) Detection of neutralizing antibody titers in mouse serum. (E–G) Flow cytometry analysis of splenocytes from immunized mice: Figure (E) represents the PBS control group, Figure (F) the mRNA vaccine group, and Figure (G) the inactivated vaccine group.
Figure 3
Figure 3
Monoclonal antibody purification and preparation of FHV. (A) SDS-PAGE analysis of five monoclonal antibodies. (B) Simulated FHV-1 infection of F81 cells and actual FHV-1 infection of F81 cells. (C) At 24 h post infection with FHV-1, F81 cells showed cytopathic effects, including cell shrinkage and detachment. (D) Nucleic acid testing for FHV in infected cell supernatant. Lane 1: Positive control. Lane 2: Negative control. Lane 3: Infected cell supernatant. (E) Nucleic acid detection of FCV in cell culture supernatant. Lane 1: Positive control. Lane 2: Negative control. Lane 3: Infected cell supernatant. (F) Nucleic acid detection in infected cell supernatant. Lane 1: FPV positive control. Lane 2: Negative control. Lane 3: Infected cell supernatant (original Figures see Supplementary Material).
Figure 4
Figure 4
Monoclonal antibody binding activity assay. (A) Indirect immunofluorescence assay for detection of monoclonal antibody binding activity. (B) Western blotting assay for evaluating the binding activity of mAbs. M stands for protein Marker. Lane 1 is the F81 cell culture supernatant, and Lane 2 is the FHV viral solution (original Figures see Supplementary Material).
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