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. 2024 Aug 15;25(16):8906.
doi: 10.3390/ijms25168906.

Specific Monoclonal Antibodies against African Swine Fever Virus Protease pS273R Revealed a Novel and Conserved Antigenic Epitope

Jiajia Zhang  1   2   3   4 , Kaili Zhang  1   2   3   4 , Shaohua Sun  1   2   3   4 , Ping He  1   2   3   4 , Dafu Deng  1   2   3   4 , Pingping Zhang  1   2   3   4 , Wanglong Zheng  1   2   3   4 , Nanhua Chen  1   2   3   4 , Jianzhong Zhu  1   2   3   4
Affiliations

Specific Monoclonal Antibodies against African Swine Fever Virus Protease pS273R Revealed a Novel and Conserved Antigenic Epitope

Jiajia Zhang et al. Int J Mol Sci. .

Abstract

The African swine fever virus (ASFV) is a large enveloped DNA virus that causes a highly pathogenic hemorrhagic disease in both domestic pigs and wild boars. The ASFV genome contains a double-stranded DNA encoding more than 150 proteins. The ASFV possesses only one protease, pS273R, which is important for virion assembly and host immune evasion. Therefore, the specific monoclonal antibody (mAb) against pS273R is useful for ASFV research. Here, we generated two specific anti-pS273R mAbs named 2F3 and 3C2, both of which were successfully applied for ELISA, Western blotting, and immunofluorescence assays. Further, we showed that both 2F3 and 3C2 mAbs recognize a new epitope of N terminal 1-25 amino acids of pS273R protein, which is highly conserved across different ASFV strains including all genotype I and II strains. Based on the recognized epitope, an indirect ELISA was established and was effective in detecting antibodies during ASFV infection. To conclude, the specific pS273R mAbs and corresponding epitope identified will strongly promote ASFV serological diagnosis and vaccine research.

Keywords: African swine fever virus; diagnosis; epitope; monoclonal antibody; pS273R.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Production and identification of the pS273R recombinant protein. (A) The purified pS273R was verified by SDS-PAGE and Coomassie blue staining, with a major band of about 37 kD. (B,C) The purified pS273R protein was verified by Western blotting using the anti-His mAb (B) and the anti-ASFV positive pig serum (1:500). (C) The pET28a vector transformed bacterial protein was used as a control.
Figure 2
Figure 2
Preparation and characterization of anti-pS273R monoclonal antibodies. (A) The reactivity of mAbs was tested in pS273R protein-based indirect ELISA. The cell supernatants of hybridoma clones 2F3 and 3C2 were used as the primary antibodies, the SP2/0 cell supernatant was used as the negative control, and the serum of immunized mice was used as positive control. The dotted line denotes the P/N value of 2.1. (B) Subclasses of mAbs 2F3 and 3C2 were determined by the monoclonal antibody isotype identification kit from CELLWAY-LAB (Luoyang, China), according to the suggested protocol. (C) Measurement of the titers of ascite mAbs 2F3 and 3C2 by pS273R protein-based indirect ELISA. The dotted line denotes the P/N value of 2.1.
Figure 3
Figure 3
The specific reactivity of pS273R mAbs was analyzed by Western blotting. (A) 293T cells were transfected with pCAGGS-pS273R-2HA (lane 2) and pCAGGS vector control (lane 1). Cells were harvested and cell lysates were detected for exogenous pS273R by Western blotting with 2F3 and 3C2 ascite mAbs (1:1000) as primary antibodies. The 3C2 mAb but not 2F3 mAb recognized a non-specific ~45 kD band, suggesting that 2F3 mAb is more specific than 3C2. (B) Primary PAMs were infected with ASFV (MOI 0.1) (lane 2) and mock-infected (lane 1) for 96 h, and cell lysates were detected for endogenous pS273R by Western blotting with the ascite mAbs 2F3 and 3C2 (1:1000) as primary antibodies. The molecular weights of protein markers are indicated on the left. M, protein markers.
Figure 4
Figure 4
The specific reactivity of pS273R mAbs was analyzed with immunofluorescence. (A) 293T cells were transfected with the pCAGGS-pS273R and pCAGGS vector, respectively. Cells were fixed at 24 h post-transfection and stained with 2F3 or 3C2 ascite mAbs (1:200), together with Goat anti-mouse IgG H&L Alexa Fluor 594. Cellular nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). (B) Primary PAMs were infected with ASFV (MOI 0.1). Cells were fixed at 72 h post-infection and stained with ascite mAbs 2F3 or 3C2 (1:200), together with secondary antibody and DAPI. The boxed areas are magnified and placed on the upper-right corners of the merged images, clearly illustrating the main cytoplasmic localization of the pS273R protein.
Figure 5
Figure 5
Identification of the antigenic epitope recognized by pS273R monoclonal antibodies. (A) Schematic of the strategy for mapping the epitope. The fragments with reactivity with pS273R mAbs are marked in blue, whereas those without reactivity with the mAbs are marked in black. (B) Western blotting analysis of the critical C terminal amino acid (left) and N terminal amino acid (right) for reactivity of pS273R fragments with the two ascite mAbs 2F3 and 3C2 (1:1000). P1 and P2 were cloned into the pCAGGS-HA vector, while P3���P12 were cloned in the pEGFP-N1 vector. Here the reactivity of P8 (1–25 aa), P10 (1–24 aa), and P11 (2–273 aa), P12 (1–273 aa) is shown, with the pCAGGS-pS273R-HA and pEGFP used as controls. The reactivities of the other fragments, P1–P7 and P9, are included in Figure S1. aa is an abbreviation for amino acid. The marks of molecular weights are indicated on the left.
Figure 6
Figure 6
Conservation analysis of identified novel linear epitopes of the pS273R protein. (A) Alignment of the epitope (1MSILEKITSSPSECAEHLTNKDSCL25) in 30 representative genotype I and II ASFV strains. The red box indicates the identified conserved epitope. (B) Prediction of the pS273R structure by using PyMOL. The epitope recognized by two mAbs is displayed in pink, and shown in the front view (a) and bottom view (b), respectively.
Figure 7
Figure 7
Establishment of indirect ELISA for detecting ASFV antibodies. (A) The pS273R epitope peptide, the positive p30 peptide (ETNECTSSFET), and a non-relevant control peptide (RSVPFEYYRIRKVKV) were used for coating at a concentration of 1 μg/mL (peptides were synthesized by GeneCreate Wuhan, China). The epitope-based ELISA was tested for detection of ASFV-positive serum, with a serum dilution of 1:10. The p values of pS273R vs. negative control and p30 vs. negative control are 0.002548 and 0.000347, respectively (n = 3). (B) The amount optimization of coating epitope peptide in indirect ELISA for detection of ASFV positive serum. (C) The optimization of serum dilution in peptide indirect ELISA with peptide coating concentration at 0.625 μg/mL. (D) Detection specificity of the established epitope-based indirect ELISA. PRRSV, PEDV, PCV2, SIV, and ASFV-positive porcine sera and negative porcine serum (NC) were used. The p-value of ASFV vs. NC is 0.000157 (n = 3). The signs ** in panels (A,D) denote p < 0.01 and are statistically very significant.
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