The Generation and Characterization of Monoclonal Antibodies against the MPXV A29L Protein

Abstract

Mpox (formerly known as monkeypox) is a zoonotic disease caused by monkeypox virus (MPXV), a DNA virus belonging to the Orthopoxvirus genus, in the Poxviridae family. The disease constitutes a moderate risk to public health at the global level. The MPXV A29L protein plays a crucial role in coordinating virion assembly and facilitating important virus-host interactions. This study focused on the expression, purification, and recombinant protein synthesis of the A29L protein of MPXV using prokaryotic systems. Using hybridoma technology, we successfully generated the monoclonal antibodies (mAbs) 1E12 and 4B2, which specifically recognize the A29L protein. These mAbs were found to be suitable for use in indirect immunofluorescence assays (IFA), Western blotting, and immunoprecipitation (IP). Our investigation also revealed that mAbs 1E12 and 4B2 could detect the A27L protein, a homologous protein found in the vaccinia virus Western Reserve (VACV WR) strain, using IFA, Western blotting, and immunoprecipitation (IP). Using mAbs 1E12 and 4B2 as primary immunological probes, A27L protein expression was detected as early as 6 h postinfection with VACV WR, with increasing protein levels being observed throughout the infection. This study enhances our understanding of the protein structure and function of MPXV and contributes to the development of specific MPXV detection methods.

Keywords: A29L; VACV; monkeypox virus; monoclonal antibody.

Figure 1

The expression and purification of the recombinant A29L protein and purification of the mAbs. (A) Lane 1: amplified products of the MPXV A29L gene. Lane 2: linearized vectors obtained by PCR amplification. (B) The purification of the recombinant A29L protein identified by SDS-PAGE. (C) A Western blot analysis of the recombinant A29L protein using an anti-His mAb. (D) The identification of the purified mAbs 1E12 and 4B2.

Figure 2

The specificity and immunogenicity of the mAbs 1E12 and 4B2 against the MPXV A29L protein strain were determined by Western blotting and IFA. (A) The Western blot detection of the A29L protein’s overexpression in HEK-293T cells using mAb 1E12, mAb 4B2, and anti-Flag mAb. (B) The IFA results of mAb 1E12 and mAb 4B2 in HEK-293T cells transfected with pCAGGS-A29L. (C) The lysates of HEK-293T cells were immunoprecipitated with either mAb 1E12 (left) or mAb 4B2 (right). The precipitates were analyzed by Western blotting using antibodies against Flag.

Figure 3

The cross-reactivity of mAbs 1E12 and 4B2 against the A27L protein of VACV. (A) A comparison of the amino acid sequence similarity between MPXV A29L and VACV WR A27L. (B) The Western blot detection of mAbs. The reactivity of mAb 1E12 and mAb 4B2 with the VACV WR A27L protein in infected cells was analyzed by Western blotting. (C) The IFA detection of mAb 1E12 and mAb 4B2 in VACV WR-infected cells. The green fluorescence represents the reaction of mAb 1E12 and mAb 4B2 with A27L in different strains of VACV. (D) The detection of the IP capacities of mAb 1E12 and mAb 4B2. The VACV WR-infected cells were lysed and immunoprecipitated with either mAb 1E12 or mAb 4B2. The precipitates were analyzed by Western blotting using antibodies against A27L.

Figure 4

The detection of A27L protein expression at different time points after VACV infection. The expression of the A27L protein in VACV WR-infected cells was monitored at various time points postinfection using IFA. Scale bar, 50 μm.