Species-Specific Conservation of Linear Antigenic Sites on Vaccinia Virus A27 Protein Homologs of Orthopoxviruses

Abstract

The vaccinia virus (VACV) A27 protein and its homologs, which are found in a large number of members of the genus Orthopoxvirus (OPXV), are targets of viral neutralization by host antibodies. We have mapped six binding sites (epitopes #1A: aa 32–39, #1B: aa 28–33, #1C: aa 26–31, #1D: 28–34, #4: aa 9–14, and #5: aa 68–71) of A27 specific monoclonal antibodies (mAbs) using peptide arrays. MAbs recognizing epitopes #1A–D and #4 neutralized VACV Elstree in a complement dependent way (50% plaque-reduction: 12.5–200 µg/mL). Fusion of VACV at low pH was blocked through inhibition of epitope #1A. To determine the sequence variability of the six antigenic sites, 391 sequences of A27 protein homologs available were compared. Epitopes #4 and #5 were conserved among most of the OPXVs, while the sequential epitope complex #1A–D was more variable and, therefore, responsible for species-specific epitope characteristics. The accurate and reliable mapping of defined epitopes on immuno-protective proteins such as the A27 of VACV enables phylogenetic studies and insights into OPXV evolution as well as to pave the way to the development of safer vaccines and chemical or biological antivirals.

Characterization of an In Vivo Neutralizing Anti-Vaccinia Virus D8 Single-Chain Fragment Variable (scFv) from a Human Anti-Vaccinia Virus-Specific Recombinant Library

“…Other authors characterized anti-D8 mAbs with VACV-neutralizing abilities only in the presence of complement, because complement is needed to increase the footprint of the mAbs [36]. These complement-dependent findings were also confirmed for mAbs directed to A27, A33, D8, H3, L1 [31,32], and B5 [63,66]. Schmaljohn et al [46] were the first to establish a recombinant human Fab library and select specific binding molecules to a number of VACV proteins.…”

“…On each plate, 50 µL scFv supernatant was added. The monoclonal antibody (mAb) 5B4/2F2 binding to epitope 1A [31,35] of VACV A27 was used as a positive control, whereas the parapoxvirus orf-specific mAb 3C5 [51] was applied as a negative control for the coated virus. The wells were washed five times with PBS/0.1% Tween after 2 h of incubation.…”

“…One linear epitope, which is highly conserved among OPXVs, was mapped at the C-terminus of A13 (amino acid residues (aa) 59-69) [30]. Moreover, six linear epitopes were mapped on the A27 protein of OPXVs (epitope #4: aa region 9-14, epitope complex #1A-D: between aa 26 and 39, and epitope #5: aa region 68-71) [31]. Other studies discovered four epitope groups on the A27 protein of VACV (group I: aa residues 21-40; group II: discontinuous; group III: aa residues 81-100; and group 4: aa residues 91-110) [32].…”

Mapping and characterization of G‐quadruplexes in monkeypox genomes

“…MP1 was identified in the antisense strand of the A27L coding gene. A27L possesses a reverse open‐reading frame gene that encodes 1 of the viral A‐type inclusion proteins frequently targeted for viral neutralization 53 and is responsible for environmental protection during Orthopoxvirus infections 54 . Meanwhile, MP2 was positioned inside the gene A50R that encodes the ATP‐dependent DNA ligase, responsible for repairing nicked DNA duplexes in Vaccinia virus 55 with mutations associated in new lineages of MPXV isolates 56 .…”

Characterisation of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin Library

“…To assess the neutralisation abilities of selected antibodies, a confluent monolayer of vero cells was grown in 24-well tissue culture plates. The PRT was performed as described before [55]. Plaques were counted by visual inspection.…”