Epitope Spreading Is Rarely Found in Pemphigus Vulgaris by Large-Scale Longitudinal Study Using Desmoglein 2–Based Swapped Molecules

Abstract

Epitope spreading is involved in inducing and maintaining self-reactivity. Epitope spreading in pemphigus vulgaris (PV), caused by IgG autoantibodies to desmoglein 3 (Dsg3) and Dsg1, was previously analyzed using Dsg3/Dsg1 extracellular domain-swapped molecules. However, precise identification of the responsible epitopes in each molecule by using only this method was problematic. In this study, we studied epitope spreading in PV by a novel immunoprecipitation-immunoblot method using Dsg3 (or Dsg1)/Dsg2 domain-swapped molecules, which overcomes the problems associated with the previous approaches. We analyzed the antigenic epitopes recognized by 212 sera collected from 53 PV patients at multiple disease stages. The major epitopes were present at the N-terminal region of Dsgs and were unchanged over the course of the disease in both anti-Dsg3 mucosal dominant-type PV and anti-Dsg3/Dsg1 mucocutaneous-type PV. These N-terminal epitopes were calcium dependent. Circulating antibodies in paraneoplastic pemphigus and pemphigus herpetiformis had unique epitope distributions, although the Dsg N-termini still contained the major epitopes. These results suggest that, after onset, intramolecular and intermolecular epitope spreading among extracellular domains on Dsg3 and Dsg1 is rare in PV and has no correlation with disease course.

Pathogenic Anti-Desmoglein 3 mAbs Cloned from a Paraneoplastic Pemphigus Patient by Phage Display

“…We further characterized the Dsg3 epitopes recognized by the isolated antibodies by immunoprecipitation/immunoblotting using a series of Dsg3/Dsg2 domain-swapped molecules produced using a baculovirus expression system (Ohyama et al, 2012). PNP-A1 bound to the EC3 domain of Dsg3, whereas PNP-B1 and PNP-C1 bound to the EC2 domain ( Figure 2b).…”

“…To determine the epitopes of the PNP patient, as well as the scFvs, we used the following baculovirus-produced recombinant human Dsg3/Dsg2 domain-swapped molecules tagged with an E-tag and a histidine tag: Dsg3-EC1 (1-101), Dsg3-EC2 (95-221), Dsg3-EC3 (212-328), Dsg3-EC4 (320-440), and Dsg3-EC5 (440-551; Ohyama et al, 2012). Baculovirus-infected insect cell culture supernatants containing recombinant molecules were incubated with the serum or scFvs for 30 minutes, and then immunoprecipitated with protein G Sepharose 4 Fast Flow beads (GE Healthcare Bio-Sciences) for serum or anti-HA agarose (Sigma-Aldrich, St Louis, MO) for scFvs (at 4 1C overnight with gentle rotation).…”

The Value of Anti-Desmoglein Enzyme-Linked Immunosorbent Assay in the Immunological Follow-Up of Pemphigus

“…Genetic and ethnic factors explain part of these observations [13], and other autoantibodies, such as anti-desmocollin or anti-acetylcholine receptor, are known to be involved in pemphigus pathophysiology [26,27]. Isolated cases of phenotypic and immunological transition from PV to PF or vice versa were described [28,29], but epitope spreading may not be implicated [30,31]. Furthermore, commercial Dsg ELISA kits detect only IgG autoantibodies, but IgA and IgE are known to be involved [27,32,33].…”

Setting the target for pemphigus vulgaris therapy

“…Longitudinal epitope-mapping studies of PV sera have shown that the targeted epitopes in DSG3 stay the same during active disease and relapse (63). Epitope spreading therefore appears to be a rare event, underscoring the static nature of the DSG3-reactive B cell repertoire in PV.…”

“…Based on the knowledge that the amino-terminal epitopes of DSG3 are predominantly targeted by PV autoantibodies (11,63), a tandem cyclic peptide consisting of DSG1 residues 81-87, highly homologous to the comparable DSG3 adhesive domain, was synthesized and shown to stabilize the trans-adhesive interface of both DSG3 and DSG1, thereby preventing autoantibody-mediated disruption of desmosomes (30,65). This treatment was effective in preventing keratinocyte pathology in vitro and also epidermal blistering when applied topically in vivo.…”