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. 2023 Mar 23;15(4):817.
doi: 10.3390/v15040817.

Efficient Cross-Screening and Characterization of Monoclonal Antibodies against Marek's Disease Specific Meq Oncoprotein Using CRISPR/Cas9-Gene-Edited Viruses

Man Teng  1   2   3 , Jin-Ling Liu  1   2 , Qin Luo  1   2   4 , Lu-Ping Zheng  1   2 , Yongxiu Yao  5 , Venugopal Nair  5 , Gai-Ping Zhang  6   7 , Jun Luo  1   2   3
Affiliations

Efficient Cross-Screening and Characterization of Monoclonal Antibodies against Marek's Disease Specific Meq Oncoprotein Using CRISPR/Cas9-Gene-Edited Viruses

Man Teng et al. Viruses. .

Abstract

Marek's disease (MD) caused by pathogenic Marek's disease virus type 1 (MDV-1) is one of the most important neoplastic diseases of poultry. MDV-1-encoded unique Meq protein is the major oncoprotein and the availability of Meq-specific monoclonal antibodies (mAbs) is crucial for revealing MDV pathogenesis/oncogenesis. Using synthesized polypeptides from conserved hydrophilic regions of the Meq protein as immunogens, together with hybridoma technology and primary screening by cross immunofluorescence assay (IFA) on Meq-deleted MDV-1 viruses generated by CRISPR/Cas9-gene editing, a total of five positive hybridomas were generated. Four of these hybridomas, namely 2A9, 5A7, 7F9 and 8G11, were further confirmed to secrete specific antibodies against Meq as confirmed by the IFA staining of 293T cells overexpressing Meq. Confocal microscopic analysis of cells stained with these antibodies confirmed the nuclear localization of Meq in MDV-infected CEF cells and MDV-transformed MSB-1 cells. Furthermore, two mAb hybridoma clones, 2A9-B12 and 8G11-B2 derived from 2A9 and 8G11, respectively, displayed high specificity for Meq proteins of MDV-1 strains with diverse virulence. Our data presented here, using synthesized polypeptide immunization combined with cross IFA staining on CRISPR/Cas9 gene-edited viruses, has provided a new efficient approach for future generation of specific mAbs against viral proteins.

Keywords: CRISPR/Cas9; MDV; Meq; gene editing; herpesvirus; monoclonal antibody.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Amino acid sequence alignment, hydrophilicity and molecular structure analysis of the Meq proteins. (a) Comparison of amino acid hydrophilicities of different virulence MDV−1 Meq proteins. The upper half of the baseline represents the hydrophilic regions. The green curve represents the Md5 strain used as reference. The blue, black and red curves represent the amino acid difference region of GX0101, 648A and GA strains relative to Md5 strain, respectively. (b) Amino acids point mutation analysis of the Meq proteins of four MDV−1 strains with different virulence. Blue and pink indicate the mutant amino acids of different strains. (c) The genomic location and domain diagram of MDV−1 Meq protein. The relative positions of synthetic polypeptides are shown by shaded grey boxes with red brackets.
Figure 2
Figure 2
Cross screening and identification of Meq-specific antibodies by immunofluorescence assay. Plaques produced in CEFs by the infection with parental or Meq-deleted viruses were stained with candidate Meq antibodies and visualized by immunofluorescence microscope. Bright field, images of MDV plaques under regular light; IFA, immunofluorescence assay. (Scale bar = 50 µm).
Figure 3
Figure 3
Staining of the Meq proteins overexpressed in 293T cells by immunofluorescence assay. The 293T cells transfected with the recombinant or empty plasmids, pEGFP-N1-Meq (Meq+) and pEGFP-N1 (Meq-), were fixed at 48 h post transfection, and then incubated with the candidate Meq antibodies and DyLight 594 labeled Goat Anti-Mouse IgG sequentially. The anti-Meq mAb FD7 served as a positive control. EGFP, enhanced green fluorescent protein with auto-fluorescence in green; Meq, MDV−1 specific oncoprotein stained in red; Merge, merged image in yellow. (Scale bar = 50 µm).
Figure 4
Figure 4
Confocal analysis of the Meq and gB proteins in MDV-infected CEFs and transformed MSB-1 cells. (a) Staining of the Meq and gB proteins in virus-infected CEF cells. The GX0101-infected CEFs were trypsinized, diluted and transferred to a special confocal culture dish. Then, the cells were fixed and incubated with Meq-specific antibodies and DyLight 594 labeled Goat Anti-Mouse IgG sequentially, followed by the incubation with anti-gB mAb HB3 and DyLight 488 labeled Goat Anti-Mouse IgG, respectively. The DAPI was used to stain and indicate the nuclei of CEF cells. Meq, MDV−1 oncoprotein stained in red; gB, MDV−1 glycoprotein B stained in green; DAPI, 4’,6-diamidino-2-phenylindole used to indicate the nuclei of cells in blue; Merge, merged image in purple; Enlarge, enlarged pictures from white brackets in merged images. (Scale bar = 20 µm or 5 µm) (b) Staining of the Meq and gB proteins in MSB-1 cells. The cells were adhered to the confocal special cell culture dish and the IFA staining was performed in the same way. Captions are the same as described above. (Scale bar = 50 µm or 10 µm).
Figure 5
Figure 5
Cross reactions of mAbs 2A9-B12 and 8G11-B2 to the viral plaques produced by different types of MDVs detected by IFA staining. The reaction spectrums of newly developed Meq mAbs to different types of MDV and HVT were determined by IFA staining. The anti-Meq mAb FD7 served as a positive control. Bright field, images of virus plaques under regular light; IFA, immunofluorescence assay. (Scale bar = 50 µm).
Figure 6
Figure 6
Western blot analysis of reactivity of mAbs 2A9-B12 and 8G11-B2 to the Meq proteins from MDV strains with different virulence. The reaction spectrums of newly developed Meq mAbs to different types of MDV and HVT were determined by Western blot analysis. Meq, MDV−1 oncoprotein. The chicken β-actin was used as protein loading control. M, protein molecular weight marker.
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