Identification of immunodominant Helicobacter pylori proteins with reactivity to H. pylori-specific egg-yolk immunoglobulin

Abstract

The importance of hens eggs as a source of specific antibodies (IgY) is well recognized. The protective effect of IgY obtained from hens immunized with Helicobacter pylori whole-cell lysate has been reported for the control of H. pylori infection. However, IgY produced by whole-cell lysates presents the possibility of cross-reactivity with other bacteria, including the normal human flora, and this could decrease the efficiency of IgY. In the present study, the immunodominant proteins of H. pylori with reactivity to H. pylori-specific IgY (IgY-Hp) were identified. IgY obtained from hens immunized with various fractions of H. pylori proteins was isolated and purified, titres of IgY-Hp against H. pylori were determined and cross-reactivity between IgY-Hp and normal human bacteria was examined by Western blot analysis. Finally, immunodominant H. pylori proteins were identified by LC/MS analysis. IgY obtained 2 months after immunization with H. pylori whole-cell lysate showed the highest antibody titre. Five immunodominant proteins were identified that were strongly reactive to IgY-Hp: urease â-subunit (62 kDa), heat-shock protein 60 (60 kDa), urease AE-subunit (26 kDa), probable peroxiredoxin (22 kDa) and probable thiol peroxidase (18 kDa). Immunization of hens with the immunodominant proteins identified would produce a more specific IgY against H. pylori.

Production of anti-Helicobacter pylori urease-specific immunoglobulin in egg yolk using an antigenic epitope of H. pylori urease

“…To address this problem, we identified the following H. pylori proteins as those that reacted immunodominantly to IgY-Hp: UreA, UreB, heatshock proteins (HSP60), probable peroxiredoxin and probable thiol peroxidase (Shin et al, 2003). Of the five immunodominant proteins, urease plays a pivotal role in the pathogenesis of H. pylori infection by protecting the bacteria from the acid environment of the stomach, promoting colonization and inducing the production of ammonia (Bury-Mone et al, 2001;Graham et al, 1992).…”

“…We previously reported that IgY-Hp was immunodominantly reactive to UreA and UreB (Shin et al, 2003). Using hydropathy plots (Kyte-Doolittle) that covered the entire sequence of H. pylori urease, we selected regions with high hydrophilicity.…”

“…IgY-HpU was isolated and purified as previously described (Shin et al, 2003). Egg yolks were separated from the whites, and the yolk preparation was mixed with an equal volume of distilled water for 30 min, followed by the addition of 0 .…”

“…However, many problems remain with regard to clinical applications, such as the crossreactivity to normal flora and the mass-production of vaccine. IgY produced using whole-cell lysates presents the likelihood of cross-reactivity with other bacteria, including the normal human flora (Shin et al, 2003), which could decrease the efficacy of IgY-Hp. Thus, immunization using a selective antigen is required.…”

“…Also, to mass-produce IgY-Hp using a selective antigen as the immunogen, mass culturing of H. pylori or recombinant DNA techniques are needed, but these processes are time-consuming and labour-intensive. In a previous study, we elucidated the five immunodominant H. pylori proteins that react strongly with IgY-Hp to address the limitations of IgY-Hp (Shin et al, 2003). Among the immunodominant proteins identified was urease, a wellknown critical virulence factor (Bury-Mone et al, 2001) and the antigen most often studied in vaccine trials.…”