Mechanistic Insights into the Neutralization of Cytotoxic Abrin by the Monoclonal Antibody D6F10

Abstract

Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74–123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1∶10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin’s toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.

Truncated abrin A chain expressed inEscherichia coli: A promising vaccine candidate

“…Additionally, tATA 4 induced improved protection as compared tATA 1 in immunized mice. This result is consistent with recent findings that the helix that is composed of amino acids 148-167 is the core of the ATA structure and truncation of the helix might destabilize the structure and result in less protective efficacy as reported by Bagaria, et al 22 This could explain why tATA 4 induced improved protection as compared tATA 1 .…”

A monoclonal antibody to an abrin chimera recognizing a unique epitope on abrin A chain confers protection from abrin-induced lethality

“…neutralizing epitope on abrin has been reported. 14 This was found to be in proximity to the active site cleft of the A subunit and hence the antibody to this epitope was observed to interfere with the enzymatic activity of abrin.…”

“…HeLa (adherent culture) and Jurkat (suspension culture) cell lines were taken for this study. The choice of the two was based on the studies carried out earlier in our laboratory to understand the mechanism of abrin neutralization by mAb D6F10 14 and the signaling triggered by abrin. 7 As shown in Figure 2 the percentage of the apoptotic population in the samples treated with abrin along with mAb A7C4 was negligible compared to the controls in both cell lines demonstrating the ability of mAb A7C4 to rescue cells from abrin-mediated cytotoxicity.…”

“…13 This mAb was found to inhibit the enzymatic activity of the abrin A chain in vitro and also conferred protection to mice in vivo. 13,14 Toward mapping the epitope corresponding to this mAb, at first various overlapping truncated forms of the abrin A chain were constructed and examined for binding to the mAb. We could conclude that the epitope lay in the amino acids 1-123 of ABA.…”

“…14 The recombinant construct retained the folding domains of ABA, enabling us to carry out mutagenesis studies to identify the key amino residues. Since the chimera included the epitope corresponding to the mAb D6F10 it was explored as a potential vaccine candidate (unpublished data).…”