Harald G. Wiker

Comparison of Antigen-Specific T-Cell Responses of Tuberculosis Patients using Complex or Single Antigens of Mycobacterium tuberculosis

We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon‐γ (IFN‐γ) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT‐6, nMPT59, nMPT64 and nMPB70) and somatic‐derived (rGroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole‐cell M. tuberculosis, M. tuberculosis culture filtrate (MT‐CF) and cell wall antigens, as well as the vaccine strain, Mycobacterium bovis bacillus Calmette–Guérin (BCG). In addition, M. tuberculosis and MT‐CF‐induced T‐cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T‐cell lines tested for antigen‐induced proliferation and IFN‐γ secretion showed that the most frequently recognized antigen was ESAT‐6, followed by MPT59, GroES, MPB70, MPT64, DnaK, GroEL and PstS. The frequency of ESAT‐6 responders, as measured both by proliferation (18/19) and secretion of IFN‐γ (16/19) was comparable to the results obtained with whole‐cell M. tuberculosis, MT‐CF and M. bovis BCG. We also observed that most of the high responders to complex antigens recognized all of the antigens tested (covariation), demonstrating that the repertoire of human T‐cell specificities induced by natural infection is directed towards several unrelated culture filtrate as well as somatic‐derived protein antigens. In conclusion, the results obtained suggest that the cellular immune response in humans is directed against several important target antigens of M. tuberculosis and that some antigens, such as ESAT‐6, are recognized by a high number of individuals. Such antigens represent candidates to be used for development of specific diagnostic reagents or in subunit vaccines.

DNA Injection in Combination with Electroporation: a Novel Method for Vaccination of Farmed Ruminants

Injection of plasmid DNA encoding antigens into rodents followed by electroporation improved the immune response when compared with injection without electroporation (Widera et al. J Immunol 2000;164:4635–40; Zucchelli et al. J Virol 2000;74:11598–607; Kadowaki et al. Vaccine 2000;18:2779–88). The present study describes the extension of this technology to farm animals, by injecting plasmid DNA encoding mycobacterial antigens (MPB70, Ag85B and Hsp65) into the muscles of goats and cattle using two different types of electrodes, both allowing DNA delivery at the site of electroporation. The animals were vaccinated under local anaesthesia without any observed immediate or long‐term distress or discomfort, or any behavioural signs of muscle damage or pathological changes after the electroporation. DNA‐injected and electroporated goats showed increased humoral response after the primary vaccination when compared with nonelectroporated animals. Improved T‐cell responses following electroporation were observed in hsp65 DNA‐vaccinated cattle. DNA injection with or without electroporation did not compromise the specificity of the tuberculin skin test. In conclusion, a protocol applying in vivo electroporation free of side effects to farmed ruminants was established. In addition, we show that DNA vaccination in combination with electroporation can improve the primary immune responses to the encoded antigens.

Definition of novel cell envelope associated proteins in Triton X-114 extracts of Mycobacterium tuberculosis H37Rv

BackgroundMembrane- and membrane-associated proteins are important for the pathogenicity of bacteria. We have analysed the content of these proteins in virulent Mycobacterium tuberculosis H37Rv using Triton X-114 detergent-phase separation for extraction of lipophilic proteins, followed by their identification with high resolution mass spectrometry.ResultsIn total, 1417 different proteins were identified. In silico analysis of the identified proteins revealed that 248 proteins had at least one predicted trans-membrane region. Also, 64 of the identified proteins were predicted lipoproteins, and 54 proteins were predicted as outer membrane proteins. Three-hundred-and-ninety-five of the observed proteins, including 91 integral membrane proteins were described for the first time. Comparison of abundance levels of the identified proteins was performed using the exponentially modified protein abundance index (emPAI) which takes into account the number of the observable peptides to the number of experimentally observed peptide ions for a given protein. The outcome showed that among the membrane-and membrane-associated proteins several proteins are present with high relative abundance. Further, a close examination of the lipoprotein LpqG (Rv3623) which is only detected in the membrane fractions of M. tuberculosis but not in M. bovis, revealed that the homologous gene in M. bovis lack the signal peptide and lipobox motif, suggesting impaired export to the membrane.ConclusionsAltogether, we have identified a substantial proportion of membrane- and membrane-associated proteins of M. tuberculosis H37Rv, compared the relative abundance of the identified proteins and also revealed subtle differences between the different members of the M. tuberculosis complex.

Bacterial proteins with cleaved or uncleaved signal peptides of the general secretory pathway

Correct protein compartmentalization is a key step for molecular function and cell viability, and this is especially true for membrane and externalized proteins of bacteria. Recent proteomic reports of Bacillus subtilis have shown that many proteins with Sec-like signal peptides and absence of a transmembrane helix domain are still observed in membrane-enriched fractions, but further evidence about signal peptide cleavage or soluble protein contamination is still needed. Here we report a proteomic screening of identified peptides in culture filtrate, membrane fraction and whole cell lysate of Mycobacterium tuberculosis. We were able to detect peptide sequencing evidence that shows that the predicted signal peptide was kept uncleaved for several types of proteins such as mammalian cell entry (Mce) proteins and PE or PE-PGRS proteins. Label-free quantitation of all proteins identified in each fraction showed that the majority of these proteins with uncleaved signal peptides are, indeed, enriched in the Triton X-114 lipid phase. Some of these proteins are likely to be located in the inner membrane while others may be outer membrane proteins.

Identification and HLA Restriction of Naturally Derived Th1-Cell Epitopes from the Secreted Mycobacterium tuberculosis Antigen 85B Recognized by Antigen-Specific Human CD4+ T-Cell Lines

ABSTRACT Antigen 85B (Ag85B/MPT59) is a major secreted protein fromMycobacterium tuberculosis which is a promising candidate antigen for inclusion in novel subunit vaccines against tuberculosis (TB). The present study was undertaken to map naturally derived T-cell epitopes from M. tuberculosis Ag85B in relation to major histocompatibility complex (MHC) class II restriction. Antigen-specific CD4+ T-cell lines were established from HLA-typed TB patients and Mycobacterium bovis BCG vaccinees by stimulation of peripheral blood mononuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-γ) secretion in response to 31 overlapping synthetic peptides (18-mers) covering the entire sequence of the mature protein. The results showed that the epitopes recognized by T-cell lines from TB patients were scattered throughout the Ag85B sequence whereas the epitopes recognized by T-cell lines from BCG vaccinees were located toward the N-terminal part of the antigen. The T-cell epitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 to 36), and p11 (aa 91 to 108) were frequently recognized by antigen-specific T-cell lines from BCG vaccinees in both proliferation and IFN-γ assays. MHC restriction analysis demonstrated that individual T-cell lines specifically recognized the complete Ag85B either in association with one of the self HLA-DRB1,DRB3, or DRB4 gene products or nonspecifically in a promiscuous manner. At the epitope level, panel studies showed that peptides p2, p3, and p11 were presented to T cells by HLA-DR-matched as well as mismatched allogeneic antigen-presenting cells, thus representing promiscuous epitopes. The identification of naturally derived peptide epitopes from the M. tuberculosisAg85B presented to Th1 cells in the context of multiple HLA-DR molecules strongly supports the relevance of this antigen to subunit vaccine design.

Purification, characterization and identification of a 32 kDa protein antigen of Mycobacterium bovis BCG☆

An immunogenic protein called P32 has been purified from Sauton zinc deficient culture filtrate of Mycobacterium bovis BCG using successively hydrophobic chromatography on Phenyl-Sepharose, ion exchange on DEAE-Sephacel and molecular sieving on Sephadex G-100. The final preparation was found to be homogeneous as based on several analyses. This P32 protein was a constituent of BCG cells grown in normal conditions. It represented about 3% of the soluble fraction of a cellular extract, and appeared as the major protein released in normal Sauton culture filtrate. This protein was found to have a molecular weight of 32,000 by SDS-polyacrylamide gel electrophoresis and in molecular sieving. Its amino acid composition showed an abundance of acidic amino acids (or their amides). The NH2-terminal amino acid sequence (6 amino acids) was determined. Purified P32 was tested by various crossed immunoelectrophoresis techniques, and was shown to belong to the antigen 85 complex in the reference system for BCG antigens. It was more precisely identified as antigen 85A. The protein antigen elicited a weak delayed hypersensitivity reaction in guinea pigs sensitized with heat-killed or living BCG. No delayed hypersensitivity reaction was observed in living BCG sensitized mice, however, it induced significant amounts of gamma interferon in cultured spleen cells from BCG-sensitized mice. Moreover, P32 either pure or as part of BCG soluble extract promoted substantial antibody levels when injected in rabbits.

Reduced expression of antigenic proteins MPB70 and MPB83 in Mycobacterium bovis BCG strains due to a start codon mutation in sigK

Mycobacterium bovis Bacille Calmette–Guérin (BCG) strains are genetically and phenotypically heterogeneous. Expression of the antigenic proteins MPB70 and MPB83 is known to vary considerably across BCG strains; however, the reason for this phenotypic difference has remained unknown. By immunoblot, we separated BCG into high‐ and low‐producing strains. By quantitative reverse transcription polymerase chain reaction (RT‐PCR), we determined that transcription of the antigen‐encoding genes, mpb70 and mpb83, follows the same strain pattern with mRNA levels reduced over 50‐fold in low‐producing strains. Transcriptome comparison of the same BCG strains by DNA microarray revealed two gene regions consistently downregulated in low‐producing strains compared with high‐producing strains, one including mpb70 (Rv2875) and mpb83 (Rv2873) and a second that includes the predicted sigma factor, sigK. DNA sequence analysis revealed a point mutation in the start codon of sigK in all low‐producing BCG strains. Complementation of a low‐producing strain, BCG Pasteur, with wild‐type sigK fully restored MPB70 and MPB83 production. Microarray‐based analysis and confirmatory RT‐PCR of the complemented strains revealed an upregulation in gene transcription limited to the sigK and the mpb83/mpb70 gene regions. These data demonstrate that a mutation of sigK is responsible for decreased expression of MPB70 and MPB83 in low‐producing BCG strains and provide clues into the role of Mycobacterium tuberculosis SigK.

A localization index for distinction between extracellular and intracellular antigens of Mycobacterium tuberculosis

A series of mycobacterial antigens were quantified by immunoelectrophoresis, enzyme-linked immunosorbent assays, or SDS-PAGE with immunoblotting using antisera against purified mycobacterial antigens. The antigens showed a characteristic distribution profile. Some had a marked quantitative dominance in the culture fluid while others had a marked dominance in sonicates of whole washed bacilli. The majority of the antigens tested could thus be located and grouped as either secreted or cytoplasmic in terms of a localization index (LI) which is described. A 5-week-old Mycobacterium tuberculosis culture fluid preparation with a low degree of lysis was valuable in the delineation of localization indexes. The various secreted antigens showed a great span in LI values, from 5 to 1000. This variation may express different degrees of secretion efficiency or differences in tendency to adhere to the bacterial surface. The identification of proteins as extracellular or cytosolic according to their LI values was in agreement for cultures of M. tuberculosis with a low degree of lysis and cultures of M. bovis BCG and M. bovis AN-5 with significant lysis of the bacterial cells.

Improved cellular and humoral immune responses against Mycobacterium tuberculosis antigens after intramuscular DNA immunisation combined with muscle electroporation

New delivery methods are needed to improve the efficiency of existing DNA vaccines. We have measured the immune response to Mycobacterium tuberculosis antigens following intramuscular DNA injection in combination with or without electroporation. Three to 6-fold increase in the number of antigen specific CD4(+) and CD8(+) T cells, measured by IFN-gamma-producing cells in an ELISPOT assay, was found in mice DNA injected and electroporated compared with non-electroporated mice. Similarly, 5 to 10-fold increase in antigen specific IgG1, IgG2a and IgG2b antibodies were found in an immunoglobulin subclass specific ELISA. A 100-fold reduction in DNA dose could be used without loss of efficiency when immunisation was combined with electroporation. A single injection of 1 microg of antigen 85b (ag85b) plasmid DNA was sufficient to elicit a higher and long lasting level of IgG2a antibodies against antigen 85B (Ag85B) compared to standard BCG vaccination. We conclude that DNA immunisation in combination with electroporation can significantly improve the immunogenicity of plasmid-based DNA vaccines.

Purification and characterization of two protein antigens from the heterogeneous BCG85 complex in Mycobacterium bovis BCG

The heterogeneous BCG85 complex is a major component of BCG culture fluid. BCG85A and BCG85B were purified by combining ammonium sulphate precipitation with chromatography on hydroxyapatite, DEAE-Sephacel and phenyl-Sepharose columns. Twenty percent of BCG85B was recovered. The chromatographic separation procedures were monitored by fused rocket immunoelectrophoresis. The BCG85 complex was found to consist of three antigens, which were heterogenous with regard to electrophoretic mobility, molecular weight (MW), hydrophobic and immunological properties. They were designated A, B and C in increasing order, according to their electrophoretic mobilities. Thus BCG85A had the lowest electrophoretic mobility, BCG85C the highest. The MW of BCG85A was found to be 31,000, while BCG85B had a slightly lower MW, 29,000, as determined by SDS-PAGE. The antigenic relationship between the components was evaluated by crossed immunoelectrophoresis and double diffusion, and reactions of partial identity between the antigens were found. The BCG85 complex occurs in far lower concentration in sonicates of BCG than in culture fluid.