The structural basis of Salmonella A2B5 toxin neutralization by antibodies targeting the glycan-receptor binding subunits

Abstract

Highlights d Antibodies targeting glycan-receptor binding B subunits can be split into two classes d The two classes are grouped by their epitope locations on the B subunit homopentamer d B homopentamers act similar to heteropentamers due to their skewed A subunit positioning d The two antibody classes exhibit significantly different neutralizing efficacies

Neutralization of Typhoid Toxin by Alpaca-Derived, Single-Domain Antibodies Targeting the PltB and CdtB Subunits

“…Next, we determined epitope locations recognized by 11 anti-PltB VHHs through a series of competition ELISAs. We exploited two IgG monoclonal antibodies (IgGs), TyTx1 and TyTx4, as we recently determined their precise binding sites on PltB subunits through cryogenic electron microscopy (cryo-EM) ( 20 ). TyTx1 and TyTx4 recognize specific amino acid residues located on the lateral and bottom sides of pentameric PltB subunits, respectively ( Fig.…”

“…TyTx1 and TyTx4 recognize specific amino acid residues located on the lateral and bottom sides of pentameric PltB subunits, respectively ( Fig. 2A ) ( 20 ). Each individual VHH, TyTx1, and TyTx4 were immobilized on the ELISA plates, as indicated on the top of each graph in Fig.…”

“…PltA and CdtB subunits are not shown. Adapted from reference 20 to explain the neutralizing epitopes recognized by TyTx1 and TyTx4 IgGs. (B) Comparative competition ELISAs of anti-PltB VHHs, TyTx1, and TyTx4.…”

“…Moreover, well-established genetic engineering strategies for generating multivalent VHHs targeting multiple epitopes on antigens make the engineering of multivalent VHHs straightforward ( 22 – 24 ), which is anticipated to be useful for future studies using T2E7, T2G9, and T4E5. For instance, we recently found that it is not straightforward to directly mask all 15 glycan receptor-binding pockets available per holotoxin using IgGs because of the A subunit-mediated interference with IgG binding to the laterally located epitopes on PltB subunits ( 20 ). It is anticipated that a mixture of several VHHs recognizing glycan receptor-binding pockets located on the lateral and bottom sides or a single engineered multivalent VHH can mask all 15 glycan receptor-binding pockets.…”

“…We carried out comparative epitope mapping studies for 41 VHHs by exploiting IgGs whose specific epitopes were recently determined by cryo-EM, TyTx1, TyTx4, and TyTx11 ( 6 , 20 ). The competition pattern of T2E7 over each VHH, TyTx1, and TyTx4 was similar to that of TyTx1 ( Fig.…”