Secreted proteins (the secretome) of the human pathogen Helicobacter pylori may mediate important pathogen-host interactions, but such proteins are technically difficult to analyze. Here, we report on a comprehensive secretome analysis that uses protein-free culture conditions to minimize autolysis, an efficient recovery method for extracellular proteins, and two-dimensional gel electrophoresis followed by peptide mass fingerprinting for protein resolution and identification. Twenty-six of the 33 separated secreted proteins were identified. Among them were six putative oxidoreductases that may be involved in the modification of protein-disulfide bonds, three flagellar proteins, three defined fragments of the vacuolating toxin VacA, the serine protease HtrA, and eight proteins of unknown function. A cleavage site for the amino-terminal passenger domain of VacA between amino acids 991 and 992 was determined by collision-induced dissociation mass spectrometry. Several of the secreted proteins are interesting targets for antimicrobial chemotherapy and vaccine development.The widespread human pathogen Helicobacter pylori is a major cause of gastric and duodenal ulcers and gastric cancer (48, 53). To identify factors of H. pylori that are potentially involved in pathogen-host interactions (11), proteome analysis has been successfully used by numerous groups (4,7,14,17,21,22,24,29,37,50). Secreted proteins (the secretome) may be of special importance, since these proteins come into direct contact with host compartments; however, technical difficulties have led to somewhat contradictory results and have prevented a comprehensive analysis (8,19,25,34,42,51). H. pylori is commonly cultivated in rich media complemented with various additions of serum containing numerous foreign proteins that are difficult to resolve from extracellular H. pylori proteins. Only a few studies have used protein-free media (1,19,26,38,49,51), and growth is usually much slower in such media. Moreover, H. pylori is particularly prone to spontaneous autolysis (34), resulting in the nonspecific release of numerous proteins; the latter makes the interpretation of protein patterns obtained from culture supernatants difficult.In this study, we optimized culture conditions for minimal autolysis, adapted a precipitation method for the optimal recovery of extracellular proteins, resolved the various secreted proteins by two-dimensional gel electrophoresis, and identified 26 protein species. Based on a comparison of the intensities of staining of specific protein species in supernatants and wholecell samples, we obtained a semiquantitative estimate for secretion selectivity. Among the secreted proteins were several redox-active enzymes, various components of the flagellar apparatus, three fragments of the vacuolating cytotoxin VacA, the serine protease and chaperone HtrA, and several previously uncharacterized proteins that are potential targets for therapy and vaccine development. To our knowledge, this is the first comprehensive analysis of the H. pylor...

Helicobacter pylori is a widespread human pathogen that can cause gastric ulcers and cancer. To identify surface proteins that may play a role in pathogen-host interactions and represent potential targets for the control of this infection, we selectively biotinylated intact H. pylori with the hydrophilic reagent sulfosuccinimidyl-6-(biotinamido)-hexanoate and purified the labeled proteins by membrane isolation, solubilization, and affinity chromatography. After separation of 82 biotinylated proteins on two-dimensional gels, 18 were identified with comparison to proteome data and peptide mass fingerprinting. Among the identified proteins, 9 have previously been shown to be surface-exposed, 7 are associated with virulence, and 11 are highly immunogenic in infected patients. In conclusion, this generally applicable combined proteome approach facilitates the rapid identification of promising targets for the control of H. pylori and might be applicable to numerous other human pathogens although larger biotinylation reagents might be required in some cases to prevent permeation of porin channels in the outer membrane.

A Vero cell-derived whole-virus H5N1 influenza vaccine has been shown to induce neutralizing antibodies directed against the hemagglutinin (HA) protein of diverse H5N1 strains in animal studies and clinical trials. However, neuraminidase-inhibiting (NAi) antibodies can reduce viral spread and may be of particular importance in the event of an H5N1 pandemic, where immunity due to HA antibodies is likely absent in the general population. Here we demonstrate the effective induction of NAi antibody titers after H5N1 vaccination in humans. In contrast to the immune response directed toward HA, a single vaccine dose induced a strong NAi response that was not significantly boosted by a second dose, most probably due to priming by previous vaccination or infection with seasonal influenza viruses. After 2 immunizations, seroconversion rates based on antibody titers against HA and NA were similar, indicating the induction of equally strong immune responses against both proteins by this H5N1 vaccine.

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The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory and tumor-promoting cytokine that occurs in two redox-dependent immunologically distinct conformational isoforms. The disease-related structural isoform of MIF (oxMIF) can be specifically and predominantly detected in the circulation of patients with inflammatory diseases and in tumor tissue, whereas the ubiquitously expressed isoform of MIF (redMIF) is abundantly expressed in healthy and diseased subjects. In this article, we report that cysteine 81 within MIF serves as a "switch cysteine" for the conversion of redMIF to oxMIF. Modulating cysteine 81 by thiol reactive agents leads to significant structural rearrangements of the protein, resulting in a decreased β-sheet content and an increased random coil content, but maintaining the trimeric quaternary structure. This conformational change in the MIF molecule enables binding of oxMIF-specific antibodies BaxB01 and BaxM159, which showed beneficial activity in animal models of inflammation and cancer. Crystal structure analysis of the MIF-derived EPCALCS peptide, bound in its oxMIF-like conformation by the Fab fragment of BaxB01, revealed that this peptide adopts a curved conformation, making the central thiol protein oxidoreductase motif competent to undergo disulfide shuffling. We conclude that redMIF might reflect a latent zymogenic form of MIF, and formation of oxMIF leads to a physiologically relevant, i.e., enzymatically active, state.

The timely development of safe and effective vaccines against avian influenza virus of the H5N1 subtype will be of the utmost importance in the event of a pandemic. Our aim was first to develop a safe live vaccine which induces both humoral and cell-mediated immune responses against human H5N1 influenza viruses and second, since the supply of embryonated eggs for traditional influenza vaccine production may be endangered in a pandemic, an egg-independent production procedure based on a permanent cell line. In the present article, the generation of a complementing Vero cell line suitable for the production of safe poxviral vaccines is described. This cell line was used to produce a replication-deficient vaccinia virus vector H5N1 live vaccine, dVV-HA5, expressing the hemagglutinin of a virulent clade 1 H5N1 strain. This experimental vaccine was compared with a formalin-inactivated whole-virus vaccine based on the same clade and with different replicating poxvirus-vectored vaccines. Mice were immunized to assess protective immunity after high-dose challenge with the highly virulent A/Vietnam/1203/2004(H5N1) strain. A single dose of the defective live vaccine induced complete protection from lethal homologous virus challenge and also full cross-protection against clade 0 and 2 challenge viruses. Neutralizing antibody levels were comparable to those induced by the inactivated vaccine. Unlike the whole-virus vaccine, the dVV-HA5 vaccine induced substantial amounts of gamma interferon-secreting CD8 T cells. Thus, the nonreplicating recombinant vaccinia virus vectors are promising vaccine candidates that induce a broad immune response and can be produced in an egg-independent and adjuvant-independent manner in a proven vector system.

A multiparameter selection of Helicobacter pylori antigens for vaccine development identified 15 candidates, 6 of which are known protective antigens. Two novel antigens with low homology to other organisms (HP0231 and HP0410) were overexpressed and purified with high yields. Both confer protective immunity in the mouse Helicobacter infection model