A highly efficient indirect ELISA and monoclonal antibody established against African swine fever virus pK205R

Abstract

African swine fever (ASF) is a contagious infectious disease with high lethality which continuously threatens the global pig industry causing huge economic losses. Currently, there are no commercially available vaccines or antiviral drugs that can effectively control ASF. The pathogen of ASF, ASF virus (ASFV) is a double-stranded DNA virus with a genome ranging from 170 to 193 kb and 151 to 167 open reading frames in various strains, which encodes 150-200 proteins. An effective method of monitoring ASFV antibodies, and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, pK205R of ASFV was successfully expressed in mammalian cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified pK205R was established and optimized. The monoclonal antibody (mAb) against pK205R recognized a conservative linear epitope (²VEPREQFFQDLLSAV¹⁶) and exhibited specific reactivity, which was conducive to the identification of the recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing pK205R. The ELISA method efficiently detected clinical ASFV infection and revealed good application prospects in monitoring the antibody level in vivo for recombinant PRRSV live vector virus expressing the ASFV antigen protein. The determination of the conserved linear epitope of pK205R would contribute to further research on the structural biology and function of pK205R. The indirect ELISA method and mAb against ASFV pK205R revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF.

Keywords: 293i cells; ASFV pK205R; epitope; indirect ELISA; recombinant PRRSV.

Figure 1

The recombinant pK205R was successfully expressed in 293i cells. (A) PCR amplification of K205R gene. (B, C) Identification of pK205R expression and purification in the culture supernatants and ultrasonic cells by SDS-PAGE (B) and WB (C). (D) Identification of purified pK205R by WB using an ASFV-positive serum as primary antibody.

Figure 2

The anti-pK205R mAb (2H11) specifically recognized 293i suspension cells transiently transfected with K205R-expressing plasmid. (A) Identification of the mAb subtypes and. (B) Identification of antibody titer of the purified 2H11 by the ELISA method. (C) 293T cells were transfected pcDNA3.4-K205R. Cells were fixed at 24 h post-transfection and immunostained with 2H11 and FITC-conjugated goat anti-mouse IgG. Cellular nuclei were counterstained with 1 μg/ml of 4′,6′-diamidino-2-phenylindole (DAPI). (D) WB assay was conducted as treated in (C) to show the reactivity of 2H11.

Figure 3

2H11 recognized specific linear B-cell epitope ²VEPREQFFQDLLSAV¹⁶. (A) Schematic diagram of K205R gene truncated fragments. (B-D) A series of truncated pK205R were constructed to pCold-TF and successfully expressed in E coli BL21 (DE3) cells. 2H11 was used to detect the truncated pK205R by WB using anti-His tag antibody or 2H11 as primary antibody, respectively. (E) Ten different truncated peptides were synthesized and tested by ELISA to show the minimum epitope recognized by 2H11.

Figure 4

2H11 recognized the conserved epitope of ASFVpK205R. (A) The percent identity of pK205R among the 25 representative ASFV strains was analyzed using MEGA. (B) Alignment analysis of the epitope (²VEPREQFFQDLLSAV¹⁶) in 25 representative ASFV strains. (C) Prediction of the pK205R structure using PyMOL. The epitope recognized by 2H11 is displayed in red color.

Figure 5

2H11 specifically recognized the recombinant PRRSV expressing pK205R. (A) The schematic representation of recombinant PRRSV virus expressing ASFV pK205R construction. (B, C) CPE and plaque morphology investigation. MARC-145 cells were infected with vA-ASFV-K205R and vHuN4-F112 (MOI = 0.001). The mock control represented non-infected MARC-145 cells. The MARC-145 cells were monitored or stained with crystal violet at 3 days post-infection. (D, E) Growth characteristics of vA-ASFV-K205R and vHuN4-F112 were evaluated in MARC-145 cells (D) and PAMs (E). (F) MARC-145 cells were infected by vA-ASFV-K205R and vHuN4-F112. WB analysis of cell lysates using an anti-N and anti-pK205R antibody. (G, H) IFA against the N protein or pK205R in MARC-145 cells (G) and PAMs (H) at 36 hpi with vA-ASFV-K205R and vHuN4-F112 (MOI = 0.1). Scale bar = 100 μm.

Figure 6

MARC-145 cells were infected by the recombinant virus vA-ASFV-K205R (P5, P10, P15, and P20 viral stocks). Cell culture supernatants were collected at 48 h post infection. (A) Sequence alignments to show the genetic stability of ASFV K205R gene (P5, P10, P15, and P20 viral stocks). The sequences of foreign gene were displayed in blue color. (B) The copy levels of K205R gene were detected in the recombinant PRRSVs (P5, P10, P15, and P20 viral stocks) by RT-qPCR. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was analyzed using t-tests. ns, not significant. (C) The pK205R expression in the recombinant PRRSVs (P5, P10, P15 and P20 viral stocks) was detected using 2H11 by IFA.

Figure 7

The indirect ELISA method against pK205R efficiently detected clinical samples and recombinant PRRSV virus expressing ASFV antigen. (A) Sensitivity testing of the ELISA method and the commercial ELISA kit using positive sera of ASFV. (B) Specificity testing of the method using CSFV, PRV, PRRSV, PEDV, FMDV, PCV2 and ASFV sera samples. (C) The PRRSV-specific humoral immune response was assessed by the S/P value identified from serum samples collected at the indicated time points from piglets in vA-ASFV-K205R, vHuN4-F112, and mock groups. (D) pK205R-specific serum antibodies of the three groups were tested using the indirect ELISA method.