A 21-35 kDa Mixed Protein Component from Helicobacter pylori Activates Mast Cells Effectively in Chronic Spontaneous Urticaria.

BACKGROUND: Helicobacter pylori (H. pylori) seem to involve in the etiology of chronic spontaneous urticaria (CSU). But studies of the pathogenic mechanism are very little. METHODS: In this study, we detected the serum-specific anti-H. pylori IgG and IgE antibodies in 211 CSU and 137 normal subjects by enzyme-linked immunosorbent assay (ELISA), evaluated the direct activation effects of H. pylori preparations and its protein components on human LAD2 mast cell line in vitro, and analyzed the specific protein ingredients and functions of the most effective H. pylori mixed protein component using liquid chromatography-mass spectrometry and ELISA assay. RESULTS: In CSU patients, the positive rate of anti-H. pylori IgG positive rate was significantly higher than that in normal controls, and the anti-H. pylori IgE levels had no statistical difference between H. pylori-infected patients with and without CSU. Further studies suggested that H. pylori preparations can directly activate human LAD2 mast cell line in a dose-dependent manner and its most powerful protein component was a mixture of 21-35 kDa proteins. Moreover, the 21-35 kDa mixed protein component mainly contained 23 kinds of proteins, which can stimulate the release of histamine, TNF-a, IL-3, IFN-γ, and LTB4 by LAD2 cells in a dose-dependent or time-dependent manner. CONCLUSIONS: A 21-35 kDa mixed protein component should be regarded as the most promising pathogenic factor contributing to the CSU associated with H. pylori infection.

Intranasal immunization with immunodominant epitope peptides derived from HpaA conjugated with CpG adjuvant protected mice against Helicobacter pylori infection.

HpaA is considered to be an effective protective antigen for vaccination against Helicobacter pylori (H. pylori) infection. Oral immunization with HpaA significantly decreases bacterial colonization in H. pylori infected mice. In this study, we investigated whether subcutaneous or intranasal immunization with HpaA could protect against H. pylori infection. Mice immunized subcutaneously with HpaA in Complete Freund's adjuvant, but not mice intranasally immunized with HpaA in CpG adjuvant acquired protection against H. pylori infection. However, intranasal immunization with immunodominant epitope peptides in CpG adjuvant protected mice against H. pylori infection, and immunodominant epitope peptides stimulated stronger Th1 responses and mediated more robust protection against H. pylori infection than subdominant ones. Our results suggest that the length of a candidate antigen is critical for particular vaccination routes, and that immunodominant epitope peptides are promising candidates for protection against H. pylori infection through nasal vaccination.

Multiple B-cell epitope vaccine induces a Staphylococcus enterotoxin B-specific IgG1 protective response against MRSA infection.

No vaccine against methicillin-resistant Staphylococcus aureus (MRSA) has been currently approved for use in humans. Staphylococcus enterotoxin B (SEB) is one of the most potent MRSA exotoxins. In the present study, we evaluated the efficacy and immunologic mechanisms of an SEB multiple B-cell epitope vaccine against MRSA infection. Synthetic overlapping peptide ELISA identified three novel B-cell immunodominant SEB epitopes (in addition to those previously known): SEB31-48, SEB133-150, and SEB193-210. Six B-cell immunodominant epitopes (amino acid residues 31-48, 97-114, 133-150, 193-210, 205-222, and 247-261) were sufficient to induce robust IgG1/IgG2b-specific protective responses against MRSA infection. Therefore, we constructed a recombinant MRSA SEB-specific multiple B-cell epitope vaccine Polypeptides by combining the six SEB immunodominant epitopes and demonstrated its ability to induce a robust SEB-specific IgG1 response to MRSA, as well as a Th2-directing isotype response. Moreover, Polypeptides-induced antisera stimulated synergetic opsonophagocytosis killing of MRSA. Most importantly, Polypeptides was more effective at clearing the bacteria in MRSA-infected mice than the whole SEB antigen, and was able to successfully protect mice from infection by various clinical MRSA isolates. Altogether, these results support further evaluation of the SEB multiple B-cell epitope-vaccine to address MRSA infection in humans.

Fine-mapping of immunodominant linear B-cell epitopes of the Staphylococcus aureus SEB antigen using short overlapping peptides.

Staphylococcal enterotoxin B (SEB) is one of the most potent Staphylococcus aureus exotoxins (SEs). Due to its conserved sequence and stable structure, SEB might be a good candidate antigen for MRSA vaccines. Although cellular immune responses to SEB are well-characterized, much less is known regarding SEB-specific humoral immune responses, particularly regarding detailed epitope mapping. In this study, we utilized a recombinant nontoxic mutant of SEB (rSEB) and an AlPO4 adjuvant to immunize BALB/c mice and confirmed that rSEB can induce a high antibody level and effective immune protection against MRSA infection. Next, the antisera of immunized mice were collected, and linear B cell epitopes within SEB were finely mapped using a series of overlapping synthetic peptides. Three immunodominant B cell epitopes of SEB were screened by ELISA, including a novel epitope, SEB205-222, and two known epitopes, SEB97-114 and SEB247-261. Using truncated peptides, an ELISA was performed with peptide-KLH antisera, and the core sequence of the three immunodominant B cell epitopes were verified as SEB97-112, SEB207-222, and SEB247-257. In vitro, all of the immunodominant epitope-specific antisera (anti-SEB97-112, anti-SEB207-222 and anti-SEB247-257) were observed to inhibit SEB-induced T cell mitogenesis and cytokine production from splenic lymphocytes of BALB/c mice. The homology analysis indicated that SEB97-112 and SEB207-222 were well-conserved among different Staphylococcus aureus strains. The 3D crystal structure of SEB indicated that SEB97-112 was in the loop region inside SEB, whereas SEB207-222 and SEB247-257 were in the ß-slice region outside SEB. In summary, the fine-mapping of linear B-cell epitopes of the SEB antigen in this study will be useful to understand anti-SEB immunity against MRSA infection further and will be helpful to optimize MRSA vaccine designs that are based on the SEB antigen.