Immune Response to an 18-Kilodalton Outer Membrane Antigen Identifies Lipoprotein 20 as a Helicobacter pylori Vaccine Candidate

Abstract

Experiments were performed using the standardized murine model of Helicobacter pylori infection to determine the immunogenicity of H. pylori outer membrane vesicles in immune protection. These vesicles, which are naturally shed from the surface of the bacterium, induce a protective response when administered intragastrically to mice in the presence of cholera holotoxin, despite the absence of the urease enzyme and associated Hsp54 chaperonin. Immunoblotting identified a specific serum immunoglobulin G (IgG) response to an 18-kDa outer membrane protein in a significant number of immunized animals. This commonly expressed, immunodominant protein was subsequently identified as lipoprotein 20 (Lpp20). Hybridoma backpacks secreting an IgG1 subclass monoclonal antibody to Lpp20 were generated in H. pylori-infected mice and were found to significantly reduce bacterial numbers, providing evidence that this surface-exposed antigen is a true vaccine candidate and not merely an antigenic marker for successful, protective immunization.Helicobacter pylori, a bacterium which is estimated to infect more than half the world's population, is associated with peptic ulcer disease (4) and the development of gastric cancer (32). Immunization against this bacterium represents a cost-effective strategy to reduce global gastric cancer rates (5) and would also have a major impact on H. pylori-related peptic ulcer disease. H. pylori vaccine candidates identified to date include the urease enzyme (20,40,51,55) and the urease enzyme chaperonin heat shock protein A (21). Mice immunized with purified VacA cytotoxin are also protected from challenge with a Tox ϩ strain of H. pylori (48). A common factor among these three vaccine candidates is their reported association with the outer membrane of H. pylori (1,16,17,27,36,52,57). The potential of catalase as an H. pylori vaccine candidate has also been identified (58). This enzyme, which is found in both the cytosol and the periplasmic space of H. pylori (28), is also thought to be surface exposed (57). More recently, the screening of recombinant H. pylori antigens (30) has identified another five potential H. pylori vaccine candidates. These include Lpp20, a conserved H. pylori lipoprotein that is membrane associated but not surface exposed (38).In our search for candidate H. pylori vaccine antigens, we have focused on the outer membrane of the bacterium. Like many other gram-negative bacteria (reviewed in reference 25), H. pylori and Helicobacter felis shed part of their outer membrane as vesicles when grown under certain conditions (34). These outer membrane vesicles (OMV) are thought to be formed when the outer membrane of the bacterium expands faster than the underlying peptidoglycan layer, resulting in portions of the membrane blebbing off the surface of growing cells (44). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis reveals that the protein and lipopolysaccharide content of these OMV closely resembles that of a Sarkosyl-insoluble outer membrane preparati...

Tip-α (hp0596 Gene Product) Is a Highly Immunogenic Helicobacter pylori Protein Involved in Colonization of Mouse Gastric Mucosa

“…HpaA was found to be cytoplasmic and inner membrane situated, whereas Lpp20 was isolated from both the inner and the outer membrane fractions of H. pylori cells [25,40]. Further systematic examination, conducted by different methods, contradicted previous results and revealed that HpaA is a flagellar sheath protein and Lpp 20 is an outer membrane antigen released from cells inside membrane vesicles [17,21]. Thus, it is likely that the methodology used for E. coli cell fractionation is not reliable when employed for H. pylori.…”

“…On the other hand, the HpaA homologue (HP0410 gene product) was selected as seroreactive [5]. Moreover, both proteins (HP0410 and Lpp20) have an immunoprotective effect [21,45].…”

Biomarkers and diagnostic tools for detection of Helicobacter pylori

“…Although some bacterial factors such as the heat shock protein, H. pylori adhesin (HpaA), and flagella have also been identified as pathogenic determinants, understanding the function of H. pylori cellular components in the pathogenesis of gastric disorders requires further investigation (Park et al 2006). In addition, a few unidentified antigenic bands such as 18, 39.5, 33, and 34 kDa have been reported to be of good diagnostic value (Andersen and Espersen 1992;Galmiche et al 2000;Keenan et al 2000). For instance, Lin et al (2007) detected seven proteins in duodenal ulcer and gastric cancer serum samples with relative molecular masses of 53 kDa (flagellin A), 53 kDa (flagellin B), 67 kDa (molecular chaperone DnaK), 61 kDa (urease β subunit), 74 kDa (flagellar hook-associated protein (FliD)), 76 kDa (flagellar hook protein), and 51 kDa (serine protease (HtrA)) (Lin et al 2007).…”

A study of recombinant protectiveH. pyloriantigens

“…Longitudinal sections, stained with a modified MayGrunwald Giemsa stain, were scanned by full length under light microscopy. Mice were considered protected or not according to the previously report [17] .…”

“…H.pylori M r 26000 outer membrane proteinspecific antibody was produced following subcutaneous immunization of the New Zealand rabbits, while age-matched control rabbits were immunized with PBS as described previously [17] . Serum antibody specificity was determined by ELISA or immunoblotting following electrophoretic transfer of SDS-PAGE-separated (150g·L -1 acrylamide) H. pylori M r 26000 outer membrane protein to 0.45µm pore size PVDF membrane.…”