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. 2024 Nov 21;108(1):522.
doi: 10.1007/s00253-024-13361-6.

Monkeypox virus A29L protein as the target for specific diagnosis and serological analysis

Chia-Yu Liang  1 , Tai-Ling Chao  2 , Chong-Syun Chao  1 , Wang-Da Liu  3   4 , Yu-Chen Cheng  2 , Sui-Yuan Chang  5   6 , Shih-Chung Chang  7   8
Affiliations

Monkeypox virus A29L protein as the target for specific diagnosis and serological analysis

Chia-Yu Liang et al. Appl Microbiol Biotechnol. .

Abstract

The unexpected monkeypox (Mpox) outbreak has been reported in many non-endemic countries and regions since May 2022. The mutant strains of Mpox virus (MPXV) were found with higher infectivity and greater capability for sustained human-to-human transmission, posing a significant public health threat. MPXV A29L, a protein homolog of vaccinia virus (VACV) A27L, plays an important role in viral attachment to host cell membranes. Therefore, MPXV A29L is considered the diagnostic target and the potential vaccine candidate for eliciting neutralizing antibodies and protective immune responses. In response to the escalating Mpox outbreak, three monoclonal antibodies (mAbs) (2-9B, 3-8G, and 2-5H) targeting the different domains of MPXV A29L have been developed in the study. Among them, 2-5H is highly specific for MPXV A29L without exhibiting cross-reactivity with VACV A27L. The antibody pairing composed of 2-5H and 3-8G has been developed as the lateral flow immunochromatographic assay for specific detection of MPXV A29L. However, these three mAbs were unable to inhibit A29L binding to heparin column or prevent MPXV infection in the neutralization test assays. The results of the serological assays using the truncated A29L fragments as the antigens showed that the Mpox patient sera contained significantly lower levels of antibodies targeting the N-terminal 1-34 residues of A29L, suggesting that the N-terminal portion of A29L is less immunogenic upon natural infection. KEY POINTS: • MAbs 2-9B, 3-8G, and 2-5H neither interrupted A29L binding to heparin nor neutralized MPXV. • The LFIA composed of 3-8G and 2-5H can specifically distinguish MPXV A29L from VACV A27L. • Mpox patient sera contained lower levels of antibodies targeting the N-terminal portion of A29L.

Keywords: A29L protein; Lateral flow immunochromatographic assay; Monkeypox (Mpox); Monkeypox virus (MPXV); Monoclonal antibody; Serological assay.

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Conflict of interest statement

Declarations. Ethical approval: The animal experiment was approved by the IACUC of National Taiwan University (approval number: NTU-112-EL-00080) and implemented in accordance with the animal care and ethics guidelines. A written informed consent was obtained from all individual Mpox patients in the study, which was approved by the Institutional Review Board of National Taiwan University Hospital. Conflict of interest: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Immunization of mice with the recombinant MPXV A29L protein. a The pET28a plasmid containing the MPXV A29L cDNA was expressed in E. coli and purified for analysis of the purity by SDS-PAGE, coomassie staining (lane 1), and WB (lane 2). b The BALB/c mice were immunized with the purified A29L in a 2-week interval and the sera were collected at the indicated days as shown in the schematic diagram. c The collected antisera were subjected to WB for monitoring the induction of the A29L-specific antibodies. d The collected antisera were subjected to ELISA as described in the “Materials and methods” section for determination of the A29L-specific IgG titer
Fig. 2
Fig. 2
The specificity of the anti-MPXV A29L mAbs 2-9B, 3-8G, and 2-5H. a Three anti-MPXV A29L mAbs, including 2-9B, 3-8G, and 2-5H, were generated by the conventional hybridoma technology. The purity of mAbs 2-9B, 3-8G, and 2-5H was analyzed by SDS-PAGE, coomassie staining (left panel), and WB (right panel). b Alignment of the amino acid sequences of VACV A27L (Swiss-Prot: P11258.3) and MPXV A29L (GenBank: AAQ72865.1) by using the Clustal Omega program (Madeira et al. 2022). Asterisk (*) indicates positions which have a single, fully conserved residue. Colon (:) indicates conservation between groups of strongly similar properties. The residues of A29L HBD are marked in red color. c The purified A27L and A29L proteins were analyzed by SDS-PAGE, coomassie staining, and WB with anti-His tag antibody, 2-9B, 3-8G, or 2-5H, respectively
Fig. 3
Fig. 3
Characterization of the binding epitopes of 2-9B, 3-8G, and 2-5H. The truncated A29L fragments (lanes 1–6) were expressed as the His-tagged sfGFP fusion proteins (a), and further utilized for SDS-PAGE, coomassie staining (b), and WB with anti-His tag antibody (c), 2-9B (d), 3-8G (e), or 2-5H (f), respectively
Fig. 4
Fig. 4
2-9B, 3-8G, and 2-5H cannot inhibit A29L binding to heparin column. The purified A29L protein was pre-incubated with 2-9B (a), 3-8G (b), or 2-5H (c), and then loaded on the HiTrap heparin HP column (Cytiva). The mAb-A29L mixtures, flowthrough fractions (Ft), wash fractions, and bound fractions were analyzed by SDS-PAGE and coomassie staining. H, antibody heavy chain. L, antibody light chain
Fig. 5
Fig. 5
2-9B, 3-8G, and 2-5H cannot neutralize MPXV. a The MPXV virions were pre-incubated with PBS, 2-9B, 3-8G, or 2-5H (200 µg/mL or 100 µg/mL) and then added to Vero E6 cell monolayers for performing the NT assays. After incubation for 7 days at 37°C in the 5% CO2 incubator, the cells were fixed with formaldehyde and stained with crystal violet. Three independent experiments were conducted for each antibody at the designated concentration. b The MPXV virions were pre-incubated with PBS, 2-9B, 3-8G, or 2-5H (100 µg/mL) in the absence or presence of the 10% baby rabbit complements (± complement) and then added to Vero E6 cell monolayers for performing the PRNT assays. Three independent experiments were conducted for each antibody group
Fig. 6
Fig. 6
The LFIA rapid test for specific detection of MPXV A29L. The LFIA rapid test was composed of the 2-9B-conjugated (a) or 2-5H-conjugated (b) colloidal gold conjugate pad, 3-8G-coated test (T) line, and the protein G-coated control (C) line on the NC membrane. PBS, A27L protein (500 ng), or A29L protein (500 ng) was added to the sample pad for starting the LFIA rapid test, respectively. c The sfGFP or sfGFP-A29L (500 ng) was also used to examine the functional specificity of the 2-5H/3-8G LFIA rapid test. The valid C lines all clearly appeared in these rapid tests, whereas the 2-5H-conjugated colloidal gold captured in the T line was only seen while adding A29L or sfGFP-A29L to the sample pad. d The various amounts of A29L protein (100, 60, 30, 15, 7.5, 5, and 0 ng/mL) were added to the sample pads of the 2-5H/3-8G LFIA rapid tests for determining the values of the limit of detection (LOD)
Fig. 7
Fig. 7
The antibody profiles of the Mpox patient sera against the truncated A29L fragments. The truncated A29L fragments were expressed as the His-tagged sfGFP fusion proteins, and further utilized for SDS-PAGE, coomassie staining (a), and WB with anti-His tag antibody (b), or the human sera (1:500) collected from Mpox patients P1–P10 (c–l), respectively. Lane 1, sfGFP fused with the 2–20 residues of A29L (30.6 kDa). Lane 2, sfGFP fused with the 2–34 residues of A29L (32.1 kDa). Lane 3, sfGFP fused with the 2–41 residues of A29L (32.9 kDa). Lane 4, sfGFP fused with the 42–110 residues of A29L (36.7 kDa). Lane 5, sfGFP fused with the full-length A29L (41.0 kDa)
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