Setting a course for intervening in host–pathogen interactions

588 Research Update References 1 Marshall, K.W. et al. (1995) Prediction of peptide affinity to HLA DRB1*0401. J. Immunol. 154, 5927–5933 2 Hammer, J. et al. (1997) HLA class II peptidebinding specificity and autoimmunity. Adv. Immunol. 66, 67–100 3 Sturniolo, T. et al. (1999) Generation of tissuespecific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices. Nat. Biotechnol. 17, 555–561 4 Rammensee, H. et al. (1999) SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50, 213–219 5 Brusic, V. et al. (1998) Prediction of MHC-class-IIbinding peptides using an evolutionary algorithm and artificial neural network. Bioinformatics 14, 121–130 6 Callan, M.F. et al. (1998) Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein–Barr virus in vivo. J. Exp. Med. 187, 1395–1402 7 Altman, J.D. et al. (1996) Phenotypic analysis of antigen-specific T lymphocytes. 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(1999) Cutting edge: identification of novel T-cell epitopes in Lol p5a by computational prediction. J. Immunol. 163, 1725–1729 14 Cochlovius, B. et al. (2000) In vitro and in vivo induction of a Th-cell response toward peptides of the melanoma-associated glycoprotein 100 protein selected by the TEPITOPE program. J. Immunol. 165, 4731–4741 15 Meyer, A.L. et al. (2000) Direct enumeration of Borrelia-reactive CD4+ T cells ex vivo by using MHC class II tetramers. Proc. Natl. Acad. Sci. U. S. A. 97, 11433–11438 16 Kotzin, B.L. et al. (2000) Use of soluble peptide–DR4 tetramers to detect synovial T cells specific for cartilage antigens in patients with rheumatoid arthritis. Proc. Natl. Acad. Sci. U. S. A. 97, 291–296 17 Kwok, W.W. et al. (2000) HLA-DQ tetramers identify epitope-specific T cells in peripheral blood of herpes-simplex-virus-type-2-infected individuals: direct detection of immunodominant antigen-responsive cells. J. Immunol. 164, 4244–4249 18 Koelle, D.M. et al. (1998) Recognition of herpes simplex virus type 2 tegument proteins by CD4+ T cells infiltrating human genital herpes lesions. J. Virol. 72, 7476–7483 19 Kern, F. et al. (1998) T-cell epitope mapping by flow cytometry. Nat. Med. 4, 975–978 20 Hernandez, J. et al. (2000) The use of HLA A2.1/p53 peptide tetramers to visualize the impact of self-tolerance on the TCR repertoire. J. Immunol. 164, 596–602 21 Liu, C. et al. (2000) Detechon of glutamic-aciddecarboxylase-activated T cells with I-Ag7 tetramers. Proc. Natl. Acad. Sci. U. S. A. 97, 14596–14601 22 Reichstetter, S. et al. (2000) Distinct T-cell interactions with HLA class II tetramers characterize a spectrum of TCR affinities in the human antigen-specific T-cell response. J. Immunol. 165, 6994–6998 23 Iezzi, G. et al. (1998) The duration of antigenic stimulation determines the fate of naive and effector T cells. Immunity 8, 89–95 24 Fahmy, T.M. et al. (2001) Increased TCR avidity after T-cell activation: a mechanism for sensing low-density antigen. Immunity 14, 135–143 25 Tian, J. et al. (2001) The frequency of high-avidity T cells determines the hierarchy of determinant spreading. J. Immunol. 166, 7144–7157 26 Rammensee, H.G. et al. (1995) MHC ligands and peptide motifs: first listing. Immunogenetics 41, 178–228 27 Southwood, S. et al. (1998) Several common HLA-DR types share largely overlapping peptidebinding repertoires. J. Immunol. 160, 3363–3373 28 Brusic, V. et al. (1998) MHCPEP, a database of MHC-binding peptides: update 1997. Nucleic Acids Res. 26, 368–371 29 Hammer, J. et al. (1994) Precise prediction of major-histocompatibility-complex-class-II– peptide interaction based on peptide side-chain scanning. J. Exp. Med. 180, 2353–2358 30 Honeyman, M.C. et al. (1998) Neural-networkbased prediction of candidate T-cell epitopes. Nat. Biotechnol. 16, 966–969 William W. Kwok* John A. Gebe Andrew Liu Stacy Agar Nancy Ptacek Gerald T. Nepom Virginia M ason Research Center, 1201 Ninth Avenue, Seattle, WA 98101, USA. * e-m ail: bkw ok@vm research.org Juergen Hammer Vaccinom e, 106 Linden Avenue, Kearny, NJ 07032, USA. David M . Koelle Dept of M edicine/Infectious Diseases, University of Washington, Box 359690, Seattle, WA 98195-9690, USA. M eeting Report Setting a course for intervening in host–pathogen interactions Frank A.W. Verreck, René R.P. de Vries and Tom H.M . Ottenhoff The 25th Dageraad Symposium on The M olecular M echanisms of Host–Pathogen Interactions in Infectious Disease: Towards Better Intervention Strategies? was held in Enkhuizen, The Netherlands, from 29 June to 3 July 2001. A small group of scientists embarked from Enkhuizen on the clipper De Dageraad to plot a course on the latest insights into host–pathogen interactions in infectious http://im m unology.trends.com disease and tack towards better intervention strategies. They set out to discuss developments in human and pathogen genetics, research into the cell biology and immunology of host–pathogen interactions, and advances in the development of vaccines. Human and pathogen genomics Both human- and pathogen-genomesequencing projects and the development of high-throughput technologies have provided an enormous potential for studying infectious diseases, from which breakthroughs can be expected in unravelling the biological processes of disease susceptibility, pathogenicity, virulence and host specificity. The human genome project will have a major impact on the development of diagnostics, therapeutics and pharmacogenomics, through the tailoring of drug treatments 1471-4906/01/$ – see front m atter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4906(01)02049-X Research Update to individual, genetically determined differences in metabolism, resulting in increased effectiveness and reduced toxicity (G-J. van Ommen, Leiden, The Netherlands). Studies of patients with severe infections with poorly pathogenic Mycobacterium and Salmonella species have revealed that many of these patients carry (causative) genetic mutations in the type-1 cytokine signaling cascade, notably, in the genes encoding interleukin-12 p40 (IL-12 p40), IL-12 receptor β1 (IL-12Rβ1), interferon γ receptor 1 (IFN-γR1), IFN-γR2 and signal transducer and activator of transcription 1 (STAT1). The immunological and clinical phenotypes resulting from these immunodeficiencies comprise a spectrum of differential severity, and provide insights into the mechanisms controlling susceptibility versus resistance to disease (T.H.M. Ottenhoff, Leiden, The Netherlands). In vitro killing assays using macrophages from partially IFN-γR-deficient patients suffering from opportunistic mycobacterial infections, showed a stronger defect in the killing of Salmonella than Toxoplasma species in the presence of IFN-γ, suggesting that alternative killing mechanisms might be able to compensate for a deficiency of IFN-γR1 in the killing of Toxoplasma, but not Salmonella (R. Janssen, Leiden, The Netherlands). Genetic analyses revealed further, novel polymorphisms in the gene encoding IL-12Rβ1, the functional implications of which have yet to be determined (M. Hoeve, Leiden, The Netherlands). The genome of Mycobacterium tuberculosis contains approximately 4000 genes. M.J. Colston (London, UK) underlined the fact that gene deletion and/or acquisition, rather than point mutation, seems to be the predominant mechanism in the evolution of species and strain variation, based on comparative microarray analyses of Mycobacterium microti, Mycobacterium bovis bacillus Calmette–Guérin (BCG) and M. tuberculosis. In the near future, microarray techniques will expand our knowledge of the regulation of genes by pathogens under changing environmental conditions (e.g. in the host cell), as well as by the host in response to microbial challenge. For instance, increased levels of outer membrane protein A from M. tuberculosis (ompATb) gene transcripts http://im m unology.trends.com TRENDS in Im m unology Vol.22 No.11 Novem ber 2001 589 Key outcomes of the meeting • Deficiencies in type-1 cytokine signaling cascades (e.g. IL-12 and IFN-γ) identified in patients suffering from prom iscuous infections w ith mycobacteria or Salm onella, com prise a heterogeneous syndrom e, w hich reveals a spectrum of genetically controlled susceptibility to intracellular pathogens. • The genom es of mycobacteria consist of highly regulated genes that allow adaptation to various niches, for exam ple, the capacity of M . tuberculosis to sw itch its m etabolism to the use of host fatty acids as a carbon source in the intracellular environm ent. • Over-expression, or expression of a dom inant-negative m utant, of the effector m olecule of Rab7 (Cord7 or RILP) affects the subcellular localization of M . bovis BCG. • Hum an m onocyte-derived m acrophages, in contrast to dendritic cells, produce IL-10 predom inantly, and not IL-12, upon (myco)bacterial stim ulation, and prohibit the efficient differentiation of Th1 cells. • Dendritic cells infected w ith M . tuberculosis and then irradiated induce, through a cross-prim ing pathway, a stronger protection to mycobacterial challenge than vaccination w ith M . bovis BCG. • In com parison w ith M . bovis BCG, a hybrid fusion protein of Ag85B and ESAT6 from M . tuberculosis, or Ag85A as a DNA vaccine in a prim e–boost regim en w ith purified Ag85 protein or BCG, have proven to be highly effective vaccines. encoding a porin molecule were found upon culturing mycobacteria at lysosomal pH (pH 5.5) and growth of an ompATb-deficient mycobacterial strain was inhibited at pH 5.5, but not pH 7.0, compared with wild-type bacteria. Using signature transposon mutagenesis to construct a mutant library, followed by testing in a mouse model, O. Neyrolles and B. Gicquel (Paris, France) identified 13 different M. tuberculosis virulence loci, encompassing genes encoding various transporters, regulators of cell-wall synthesis, a lipase and a transcriptional regulator. ‘...alternative killing mechanisms might be able to compensate for a deficiency of IFN-γγ R1 in the killing of Toxoplasma, but not Salmonella...’ The cell biology of host–pathogen interactions D. Russell (Ithaca, NY, USA) discussed the positioning of the mycobacterial phagosome in the endosomal system of the host cell, including its lack of maturation and acidification. However, IFN-γmediated activation of macrophages results in further acidification of the phagosome (from pH 6.3 to pH 5.5) and its intersection with the HLA-DM+ biosynthetic route of the MHC-class-IIloading compartment. Intracellular mycobacteria respond to the intracellular environment by switching their metabolism to the use of host fatty acids as an alternative carbon source. An elegant approach was presented that allows enrichment for mycobacteria that are deficient in their capacity to induce phagosomal arrest by purifying these from phagolysosomes by magnetic sorting from iron–dextran-charged macrophages. Molecular analysis of these mutants should provide an insight into the factors controlling phagosomal arrest. Rab7 is a member of the small GTPase family that regulates lysosomal transport and fusion. The recent discovery of an effector molecule of Rab7, the connector of Rab7 and dynein (Cord7) or Rabinteracting lysosomal protein (RILP), illustrates, according to J. Neefjes (Amsterdam, The Netherlands), that a fundamental understanding of cellular biological mechanisms of vesicle transport and maturation will provide a better understanding of how intracellular pathogens interact with and survive in their host cells. Importantly, this will provide novel tools for the molecular manipulation of infected cells. Indeed, the over-expression of Cord7/RILP protein or transfection of a dominant-negative mutant into HeLa cells was shown to affect the subcellular localization of green fluorescent protein (GFP)-tagged M. bovis BCG (M. Marsman, Amsterdam, The Netherlands). CD1 molecules present (myco)bacterial glycolipids to T cells. Whereas CD1a lacks endosomal targeting signals and is found exclusively at the cell surface or in recycling 590 Research Update vesicles, CD1b and CD1c colocalize with mycobacteria and segregate from MHC class II by staying in the endosomal compartment upon the maturation of dendritic cells (DCs) (P. Peters and N. van der Wel, Amsterdam, The Netherlands). This differential distribution of CD1 molecules probably reflects differential antigen (Ag)-loading qualities. Induction of acquired immunity The induction of cell-mediated immunity is crucial for protection against many pathogens. C. Melief (Leiden, The Netherlands) elaborated on the signals required for the activation of Ag-presenting cells and the induction of cytotoxic T lymphocytes (CTLs). The expression of CD40 is up-regulated in DCs upon stimulation with bacterial products, and ligation of CD40 is sufficient to induce maturation of the DC and subsequently, induction of CD8+ CTL responses, thus explaining the contribution of CD40 ligand (CD40L)expressing CD4+ T cells. Similarly, signaling through CD40–CD40L interactions bypasses the inhibitory action of regulatory T cells (R. Toes, Leiden, The Netherlands). Triggering of the tumor necrosis factor receptor family member 4-1BB on CD8+ CTLs by the 4-1BB ligand expressed by DCs appeared to be crucial for the sustained expansion of CTL populations, and this interaction was only effective in the context of CD28 costimulation. ‘Intracellular mycobacteria respond to the intracellular environment by sw itching their metabolism to the use of host fatty acids as an alternative carbon source.’ M.J. Colston studied DCs as a vehicle for mycobacteria in a vaccination model and showed that they confer protection of the order of one to two logs greater than BCG vaccination. The DCs, which were infected with M. tuberculosis and, subsequently, irradiated to kill the bacteria, exerted their protective effect through a ‘crosspriming’ pathway. The finding that only human monocyte-derived DCs and not monocytederived macrophages (Mφs) produce IL-12 upon Toll-like receptor 2 (TLR2)mediated triggering by mycobacteria or TLR4-mediated triggering by lipopolysaccharide supports the notion that DCs, but not Mφs, are crucial for the http://im m unology.trends.com TRENDS in Im m unology Vol.22 No.11 Novem ber 2001 effective activation of T-cell-mediated immunity (F.A.W. Verreck, Leiden, The Netherlands). In an attempt to understand the lack of secretion of IL-12 by Mφs, it was found that Mφs, in contrast to DCs, secrete IL-10 in response to low-dose bacterial triggering. Vaccine development Although M. bovis BCG is the only available vaccine against tuberculosis (TB) to date, it has a disputable record of efficiency, ranging from minus 50% to plus 90%, in various field trials. In addition to differences in genetic susceptibility of the population, differences in vaccination strategies or exposure to UV light (B.J. Vermeer, Leiden, The Netherlands), exposure to environmental mycobacteria probably influences the outcome of vaccination. To study this, P. Andersen (Copenhagen, Denmark) established an environmental sensitization model in the mouse (by successive mycobacterial sensitization, chemotherapeutic clearance and subsequent BCG vaccination). He showed that pre-sensitization with M. avium led to a rapid killing of subsequently administered BCG, thereby nullifying its protective efficacy. Interestingly, a hybrid fusion protein containing the two immunodominant Ags of M. tuberculosis, 85B and early secretory antigenic target 6 (ESAT6), appeared to be a highly effective vaccine in both mouse and guinea pig models of TB. Importantly, the efficacy of this vaccine was not affected by environmental mycobacterial presensitization. Intramuscular vaccination with plasmid DNA encoding the highly conserved and immunogenic Ag85A from M. tuberculosis mediated protective immunity to TB in mice and in guinea pigs, as well as to infection with Mycobacterium ulcerans, the causative agent of Buruli ulcer (K. Huygen, Brussels, Belgium). Best results were obtained using a complex of DNA encoding Ag85 in the cationic lipid vaxfectin and a prime-and-boost regimen of DNA and purified Ag85 protein in adjuvant, respectively. Moreover, K. Huygen showed that prior priming of BALB/c mice with DNA encoding Ag85A could increase the potency of a BCG vaccine against an intravenous TB challenge. R. de Vries and A. Geluk (Leiden, The Netherlands), studying Mycobacteriumspecific T-cell responses, discussed the major role of HLA class I and class II molecules in dictating the Ag and epitope specificity of the anti-mycobacterial T-cell response. They described the use of HLAtransgenic mice to identify human class-Iand class-II-restricted T-cell determinants, and their potential as a preclinical vaccination model for mycobacterial diseases. Novel candidate vaccine epitopes for the induction of CD4+ and CD8+ T cells in TB were defined by A. Geluk, and a polyepitope approach was presented. J. Thole (Lelystad, The Netherlands) illustrated the applicability of genetically engineered mycobacteria for efficient vaccine delivery of selected epitopes, using delivery systems involving recombinant superoxide dismutase (to stimulate CD4+ T cells) or recombinant 19 kDa lipoprotein (to stimulate both CD4+ and CD8+ T cells). ‘...[vaccination with] M. bovis BCG...has a disputable record of efficiency, ranging from minus 50% to plus 90%, in various field trials.’ Concluding remarks Exciting new developments are taking place in the study of molecular host–pathogen interactions spurred by the pace of research in pathogen and host genomics and proteomics. This meeting focused on intracellular bacteria, in particular mycobacteria. Combined with better insights into cellular biological and immunological interactions between the human host and its pathogens, these developments are now starting to result in better vaccination formulae and strategies. Acknow ledgements The symposium was organized on behalf of the Foundation Development Fund Immunohematology (SOFI) by the Dept of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands. Financial support was received from Aventis Pasteur, KNCV Tuberculosebestrijding, the Royal Dutch Academy for Sciences (KNAW) National Reference Center for Histocompatibility, Foundation ‘De Drie Lichten’. Frank A.W. Verreck* René R.P. de Vries Tom H.M . Ottenhoff Dept of Im m unohem atology and Blood Transfusion (IHB)/E3-Q, Leiden University M edical Center, Albinusdreef 2, NL 2333ZA Leiden, The Netherlands. * e-m ail: f.a.w.verreck@lum c.nl