Identification and characterization of a common B-cell epitope on EIAV capsid proteins

Abstract

The equine infectious anemia virus (EIAV) capsid protein (p26) is one of the major immunogenic proteins during EIAV infection and is widely used for the detection of EIAV antibodies in horses. However, few reports have described the use of EIAV-specific monoclonal antibodies (MAbs) in etiological and immunological detection. Previously, we developed an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) for the quantification of the EIAV p26 protein level. However, the epitopes recognized by the MAbs were not identified, and the utilization of the MAbs needs to be evaluated. In this study, we characterized two monoclonal antibodies (9H8 and 1G11 MAbs) against EIAV p26. Two B-cell epitopes are located in amino acid residues, NLDKIAEE (HE) and KNAMRHLRPEDTLEEKMYAC (GE) for the 9H8 and 1G11 MAbs, respectively. The 1G11 epitope (GE) varied among viruses isolated worldwide but can be recognized by anti-EIAV sera from different regions, including China, the USA, and Argentina. Meanwhile, 1G11 MAb could react with the mutants of almost all the EIAV strains. Furthermore, we found that the histidine at position 204 (H204), leucine at position 205 (L205), and aspartic acid at position 209 (D209) of EIAV p26 individually played pivotal roles in binding with the 1G11 MAb. Our results revealed that the GE peptide might be a common B-cell binding epitope of EIAV antibodies. This is also the first report to identify a broad-spectrum monoclonal antibody (1G11) against p26 of EIAV. These findings may provide a useful basis for the development of new diagnostic assays for EIAV.

High Genomic Variability in Equine Infectious Anemia Virus Obtained from Naturally Infected Horses in Pantanal, Brazil: An Endemic Region Case

“…Indeed, serologically silent equines have been observed in several regions of the world [51]. In our study, both Brazilian p26 proteins displayed changes in surface amino acids in the described epitopes [39][40][41] (Figure 3). These results suggest that more studies related to gag gene characterization may contribute to better detection of EIAV infection and disease control in an endemic area (at least in the Pantanal region), where adaptation by the virus to their hosts allows the persistence of infected animals for years.…”

“…Only Miyazaki and Wyoming presented an alteration of valine to leucine or isoleucine in the active site flap (both nonpolar amino acids). The RT motifs described for HIV were also conserved in BRA1 and BRA2 (DIGD and DD) [41]. RN contained four invariant residues and a glycine-rich motif conserved in all sequences.…”

“…biochem.duke.edu/) [38]. Previously described epitope sequences [39][40][41] were compared by structure alignment using the CHIMERA program [42].…”

Equine lentivirus counteracts SAMHD1 restriction by Rev-mediated degradation of SAMHD1 via the BECN1-dependent lysosomal pathway

“…The mouse anti-Flag (F1804), mouse anti-HA (H9658), mouse anti-actin beta (ACTB; A1978) monoclonal antibodies, rabbit anti-SAMHD1 (SAB2102077), rabbit anti-Flag (F7425), and rabbit anti-HA (H6908) were purchased from Sigma-Aldrich; the rabbit anti-BECN1 (ab207612), rabbit anti-ATG3 (ab108282), rabbit anti-ATG5 (ab108327), and rabbit anti-ATG7 (ab52472) antibodies were purchased from Abcam; the rabbit anti-class III PtdIns3K (4263) and rabbit anti-LC3B (2775) antibodies were purchased from Cell Signaling Technology; and the rabbit anti-GST (10000-0-AP) antibodies were purchased from Proteintech. Monoclonal antibodies against P26, which is the capsid protein of EIAV, were prepared in our laboratory [ 62 ]. DyLight™ 800-labeled goat anti-mouse (5230–0415) and DyLight™ 680-labeled goat anti-rabbit (5230–0403) secondary antibodies were purchased from KPL.…”

Equine Myxovirus Resistance Protein 2 Restricts Lentiviral Replication by Blocking Nuclear Uptake of Capsid Protein

“…The cells were then fixed with 4% paraformaldehyde for 30 min and permeabilized in 0.1% Triton X-100 for 15 min. The cells were incubated with anti-HA or -p26 antibodies (made in-house) ( 49 ) or anti-p24 antibodies (provided by Yongtang Zheng) ( 50 ), followed by staining using secondary antibodies conjugated with fluorescein isothiocyanate (FITC) or tetramethyl rhodamine isocyanate (TRITC) (Sigma). Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, China).…”