[Novel vaccination concepts on the basis of modern insights into immunology]. / Neue Impfstoffkonzepte auf Basis moderner Erkenntnisse der Immunologie.

Since their introduction more than 200 years ago, vaccines have prevented millions of deaths caused by infectious diseases. This progress was possible because these vaccines protect through antibodies, which are relatively easily stimulated. In the meantime, we understand that diseases such as AIDS, tuberculosis, malaria and hepatitis C cannot be tackled by these conventional approaches. Recent insights into immunology provide the basis for the development of custom-tailored vaccines to successfully combat these threatening infections. These new generation vaccines comprise components that modulate the mediators of immunity (B cells, T cells, antigen-presenting cells and cytokines) in such a way that the best possible immune response develops. Alternative application methods offer the possibility to further improve the immune response. Thus, hope remains that the remarkable increase in knowledge in the areas of immunology and infectious disease research will help to successfully control infectious diseases.

Small-fragment genomic libraries for the display of putative epitopes from clinically significant pathogens.

Taking advantage of whole genome sequences of bacterial pathogens in many thriving diseases with global impact, we developed a comprehensive screening procedure for the identification of putative vaccine candidate antigens. Importantly, this procedure relies on highly representative small-fragment genomic libraries that are expressed to display frame-selected epitope-size peptides on a bacterial cell surface and to interact directly with carefully selected disease-relevant high-titer sera. Here we describe the generation of small-fragment genomic libraries of Gram-positive and Gram-negative clinically significant pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae, Enterococcus faecalis, Helicobacter pylori, Chlamydia pneumoniae, the enterotoxigenic Escherichia coli, and Campylobacter jejuni. Large-scale sequencing revealed that the libraries, which provide an average of 20-fold coverage, were random and, as demonstrated with two S. aureus libraries, highly representative. Consistent with the comprehensive nature of this approach is the identification of epitopes that reside in both annotated and putatively novel open reading frames. The use of these libraries therefore allows for the rapid and direct identification of immunogenic epitopes with no apparent bias or difficulty that often associate with conventional expression methods.

Analysis of sequence-specific binding of RNA to Hsp70 and its various homologs indicates the involvement of N- and C-terminal interactions.

Members of the 70-kDa family of molecular chaperones assist in a number of molecular interactions that are essential under both normal and stress conditions. These functions require ATP and co-chaperone molecules and are associated with a cyclic transition of intramolecular conformational changes. As a new putative function, we have previously shown that mammalian Hsp/Hsc70 as well as a distant relative, Hsp110, selectively bind certain RNA sequences via their N-terminal ATP-binding domain. To investigate this phenomenon in more detail, here we examined RNA-binding affinity and specificity of various deletion mutants of human Hsp70. We demonstrate, that, although the N-terminal ATPase domain alone is sufficient for RNA binding, its binding affinity is considerably reduced when compared to that of the full-length protein. Additionally, we provide evidence that binding of RNA to a membrane-immobilized protein partner results in complete loss of RNA sequence specificity. Using various Hsp70 homologs, we show distinct RNA-binding properties of these proteins judged by sequence specificity, ribopolymer sensitivity, and northwestern analysis. Finally, we present data disclosing that RNA binding by DnaK, the Escherichia coli homolog, is influenced by the activity of its co-chaperones, DnaJ and GrpE. We conclude that the RNA-binding capability of this class of molecular chaperones is a conserved feature and it is strongly influenced by the structural and conformational properties. Furthermore, the notion that RNA binding of some Hsp70 family members is influenced by co-chaperones suggests an RNA-binding cycle resembling the protein-binding property of the chaperones.

Defined synthetic vaccines.

Although vaccines have proven very successful in preventing certain infectious diseases, progress in the field has been slowed by the tediousness of developing classical vaccines consisting of whole pathogens. Thus, there is great need for improvement in several areas: firstly, the range of diseases which can be treated has to be expanded. Secondly, antigens have to be defined to make the use of whole pathogens as antigen obsolete. And thirdly, new adjuvants have to be developed which show low toxicity, high potency and are also able to drive the immune response in the desired direction. Ideally, a vaccine would only consist of well-characterized, synthetic materials. This review summarizes the different approaches for the development of completely defined synthetic vaccines.

Cleavage of poly(A) tails on the 3'-end of RNA by ribonuclease E of Escherichia coli.

RNase E initiates the decay of Escherichia coli RNAs by cutting them internally near their 5'-end and is a component of the RNA degradosome complex, which also contains the 3'-exonuclease PNPASE: Recently, RNase E has been shown to be able to remove poly(A) tails by what has been described as an exonucleolytic process that can be blocked by the presence of a phosphate group on the 3'-end of the RNA. We show here, however, that poly(A) tail removal by RNase E is in fact an endonucleolytic process that is regulated by the phosphorylation status at the 5'- but not the 3'-end of RNA. The rate of poly(A) tail removal by RNase E was found to be 30-fold greater when the 5'-terminus of RNA substrates was converted from a triphosphate to monophosphate group. This finding prompted us to re-analyse the contributions of the ribonucleolytic activities within the degradosome to 3' attack since previous studies had only used substrates that had a triphosphate group on their 5'-end. Our results indicate that RNase E associated with the degradosome may contribute to the removal of poly(A) tails from 5'-monophosphorylated RNAs, but this is only likely to be significant should their attack by PNPase be blocked.

Ligand-mediated protection against phage lysis as a positive selection strategy for the enrichment of epitopes displayed on the surface of E. coli cells.

We present a novel strategy, termed CISTEM, which allows direct in vivo screening of polypeptides displayed on the surface of E. coli cells by a combination of ligand-mediated protection and phage-mediated selection. The effectiveness of this new approach was demonstrated by displaying the T7.tag on the surface of E. coli as a fusion with the outer membrane protein A, the receptor for bacteriophage K3. A monoclonal T7.tag antibody was used as protective ligand for T7.tag-displaying cells and phage K3 for the elimination of unprotected cells. When populations of bacteria, containing between 6 to 10,000 cells displaying the T7.tag and approximately 10(8) cells displaying an unrelated OmpA fusion protein, were infected with phage K3, specific and antibody-dependent survival of T7.tag displaying cells was observed, yielding an enrichment factor of up to 10(7)-fold. The CISTEM technology was used to select sequences from a T7.tag-based, randomised library and the results were compared to those obtained from selection by MACS with the same library. Together, these results reveal a novel in vivo screening strategy in which an E. coli phage receptor is used as display plafform and selection is performed in suspension upon addition of a protective ligand and a bacteriophage. Extentions and modifications of the basic strategy should lead to novel applications for the identification of protein-ligand interactions.

Enhanced cleavage of RNA mediated by an interaction between substrates and the arginine-rich domain of E. coli ribonuclease E.

Endonucleolytic cutting by the essential Escherichia coli ribonuclease RNaseE has a central role in both the processing and decay of RNA. Previously, it has been shown that an oligoribonucleotide corresponding in sequence to the single-stranded region at the 5' end of RNAI, the antisense regulator of ColE1-type plasmid replication, is efficiently cut by RNaseE. Combined with the knowledge that alteration of the structure of stem-loops within complex RNaseE substrates can either increase or decrease the rate of cleavage, this result has led to the notion that stem-loops do not serve as essential recognition motifs for RNaseE, but can affect the rate of cleavage indirectly by, for example, determining the single-strandedness of the site or its accessibility. We report here, however, that not all oligoribonucleotides corresponding to RNaseE-cleaved segments of complex substrates are sufficient to direct efficient RNaseE cleavage. We provide evidence using 9 S RNA, a precursor of 5 S rRNA, that binding of structured regions by the arginine-rich RNA- binding domain (ARRBD) of RNaseE can be required for efficient cleavage. Binding by the ARRBD appears to counteract the inhibitory effects of sub-optimal cleavage site sequence and overall substrate conformation. Furthermore, combined with the results from recent analyses of E. coli mutants in which the ARRBD of RNase E is deleted, our findings suggest that substrate binding by RNaseE is essential for the normal rapid decay of E. coli mRNA. The simplest interpretation of our results is that the ARRBD recruits RNaseE to structured RNAs, thereby increasing the localised concentration of the N-terminal catalytic domain, which in turn leads to an increase in the rate of cleavage.

Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding.

The adaptation of mRNA stability to environmental changes is a means of cells to adjust the level of gene expression. The Escherichia coli ompA mRNA has served as one of the paradigms for regulated mRNA decay in prokaryotes. The stability of the transcript is known to be correlated inversely with the bacterial growth rate. Thus, the regulation of ompA mRNA stability meets the physiological needs to adjust the level of ompA expression to the rate of cell division. Recently, host factor I (Hfq/HF1) was shown to be involved in the regulation of ompA mRNA stability under slow growth conditions. Here, we present the first direct demonstration that 30S ribosomes bound to the ompA 5'-UTR protect the transcript from RNase E cleavage in vitro. However, the 30S protection was found to be abrogated in the presence of Hfq. Toeprinting and in vitro translation assays revealed that translation of ompA is repressed in the presence of Hfq. These in vitro studies are corroborated by in vivo expression studies demonstrating that the reduced synthesis rate of OmpA effected by Hfq results in functional inactivation of the ompA mRNA. The data are discussed in terms of a model wherein Hfq regulates the stability of ompA mRNA by competing with 30S ribosomes for binding to the ompA 5'-UTR.

Mammalian Hsp70 and Hsp110 proteins bind to RNA motifs involved in mRNA stability.

In this study, in vitro RNA binding by members of the mammalian 70-kDa heat shock protein (Hsp) family was examined. We show that Hsp/Hsc70 and Hsp110 proteins preferentially bound AU-rich RNA in vitro. Inhibition of RNA binding by ATP suggested the involvement of the N-terminal ATP-binding domain. By using deletion mutants of Hsp110 protein, a diverged Hsp70 family member, RNA binding was localized to the N-terminal ATP-binding domain of the molecule. The C-terminal peptide-binding domain did not bind RNA, but its engagement by a peptide substrate abrogated RNA binding by the N terminus of the protein. Interestingly, removal of the C-terminal alpha-helical structure or the alpha-loop domain unique to Hsp110 immediately downstream of the peptide-binding domain, but not both, resulted in considerably increased RNA binding as compared with the wild type protein. Finally, a 70-kDa activity was immunoprecipitated from RNA-protein complexes formed in vitro between cytoplasmic proteins of human lymphocytes and AU-rich RNA. These findings support the idea that certain heat shock proteins may act as RNA-binding entities in vivo to guide the appropriate folding of RNA substrates for subsequent regulatory processes such as mRNA degradation and/or translation.

Growth phase-regulated induction of Salmonella-induced macrophage apoptosis correlates with transient expression of SPI-1 genes.

Invasive Salmonella has been reported to induce apoptosis in a fraction of infected macrophages within 2 to 14 h from the time of infection by a mechanism involving the type III secretion machinery encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we show that bacteria in the transition from logarithmic to stationary phase cause 90% of the macrophages to undergo phagocytosis-independent, caspase-mediated apoptosis within 30 to 60 min of infection. The ability of Salmonella to induce this rapid apoptosis was growth phase regulated and cell type restricted, with epithelial cells being resistant. Apoptosis induction was also abrogated by disruption of the hilA gene (encoding a regulator of SPI-1 genes) and by the expression of a constitutively active PhoPQ. hilA itself and a subset of SPI-1 genes were transiently expressed during aerobic growth in liquid medium. Interestingly, however, hilA was found to be required only for the expression of the prgH gene, while sipB, invA, and invF were expressed in a hilA-independent manner. The expression of SPI-1 genes and the secretion of invasion-associated proteins correlated temporally with the induction of apoptosis and are likely to represent its molecular basis. Thus, growth phase transition regulates the expression and secretion of virulence determinants and represents the most efficient environmental cue for apoptosis induction reported to date.

The Epstein-Barr virus nuclear antigen 2 (EBNA2), a protein required for B lymphocyte immortalization, induces the synthesis of type I interferon in Burkitt's lymphoma cell lines.

Epstein-Barr virus nuclear antigen 2 (EBNA2), a protein involved in cell transformation, interferes with the cellular response to type I interferons (IFN-alpha/beta). We investigated the function of conditionally expressed EBNA2 in the context of the IFN response in Burkitt's lymphoma cell lines. Expression of EBNA2 led to the transcriptional activation of both endogenous or transfected IFN-stimulated genes (ISGs), genes which contain within their promoters either the interferon-stimulated response element (ISRE) or the gamma interferon activation site (GAS). In search of a molecular mechanism for the transcriptional induction of ISGs, we observed an EBNA2-dependent synthesis of IFN-beta mRNA at low levels and the secretion of low amounts of IFN. A transfected IFN-beta promoter responded to EBNA2 activation, and a sequence closely resembling a RBP-Jkappa binding site was pinpointed as a potential target of EBNA2 activity. EBNA2-dependent transcriptional induction of the IFN-beta promoter occurred in EBV-negative Burkitt's lymphoma cells, indicating that other EBV genes were not required for the induction of IFN-beta synthesis.

Salmonella typhimurium and lipopolysaccharide stimulate extracellularly regulated kinase activation in macrophages by a mechanism involving phosphatidylinositol 3-kinase and phospholipase D as novel intermediates.

Activation of the extracellularly regulated kinase (ERK) pathway is part of the early biochemical events that follow lipopolysaccharide (LPS) treatment of macrophages or their infection by virulent and attenuated Salmonella strains. Phagocytosis as well as the secretion of invasion-associated proteins is dispensable for ERK activation by the pathogen. Furthermore, the pathways used by Salmonella and LPS to stimulate ERK are identical, suggesting that kinase activation might be solely mediated by LPS. Both stimuli activate ERK by a mechanism involving herbimycin-dependent tyrosine kinase(s) and phosphatidylinositol 3-kinase. Phospholipase D activation and stimulation of protein kinase C appear to be intermediates in this novel pathway of MEK/ERK activation.

Host factor I, Hfq, binds to Escherichia coli ompA mRNA in a growth rate-dependent fashion and regulates its stability.

The stability of the ompA mRNA depends on the bacterial growth rate. The 5' untranslated region is the stability determinant of this transcript and the target of the endoribonuclease, RNase E, the key player of mRNA degradation. An RNA-binding protein with affinity for the 5' untranslated region ompA was purified and identified as Hfq, a host factor initially recognized for its function in phage Qbeta replication. The ompA RNA-binding activity parallels the amount of Hfq, which is elevated in bacteria cultured at slow growth rate, a condition leading to facilitated degradation of the ompA mRNA. In hfq mutant cells with a deficient Hfq gene product, the RNA-binding activity is missing, and analysis of the ompA mRNA showed that the growth-rate dependence of degradation is lost. Furthermore, the half-life of the ompA mRNA is prolonged in the mutant cells, irrespective of growth rate. Hfq has no affinity for the lpp transcript whose degradation, like that of bulk mRNA, is not affected by bacterial growth rate. Compatible with our results, we found that the intracellular concentration of RNase E and its associated degradosome components is independent of bacterial growth rate. Thus our results suggest a regulatory role for Hfq that specifically facilitates the ompA mRNA degradation in a growth rate-dependent manner.

The endoribonucleolytic N-terminal half of Escherichia coli RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly.

Escherichia coli RNase E, an essential single-stranded specific endoribonuclease, is required for both ribosomal RNA processing and the rapid degradation of mRNA. The availability of the complete sequences of a number of bacterial genomes prompted us to assess the evolutionarily conservation of bacterial RNase E. We show here that the sequence of the N-terminal endoribonucleolytic domain of RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria. Furthermore, we demonstrate that the Synechocystis sp. homologue binds RNase E substrates and cleaves them at the same position as the E. coli enzyme. Taken together these results suggest that RNase E-mediated mechanisms of RNA decay are not confined to E. coli and its close relatives. We also show that the C-terminal half of E. coli RNase E is both sufficient and necessary for its physical interaction with the 3'-5' exoribonuclease polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase, which are components of a "degradosome" complex. Interestingly, however, the sequence of the C-terminal half of E. coli RNase E is not highly conserved evolutionarily, suggesting diversity of RNase E interactions with other RNA decay components in different organisms. This notion is supported by our finding that the Synechocystis sp. RNase E homologue does not function as a platform for assembly of E. coli degradosome components.

RNase E, the major player in mRNA degradation, is down-regulated in Escherichia coli during a transient growth retardation (diauxic lag).

The endoribonuclease RNase E plays a major part in mRNA degradation in Escherichia coli in addition to its role in processing rRNA. RNase E is encoded by an essential gene, rne, also known as ams and hmp, which is autoregulated post-transcriptionally. Here we report a transient decrease in the steady state level of the full-length rne transcript and a corresponding decline in the amount of the protein and enzymatic activity. During this period an mRNA fragment, lacking an intact 5' end, accumulates. This down-regulation of RNase E occurs under aerobic growth conditions in rich medium during a short diauxic lag in mid-exponential phase; it most likely reflects an exhaustion of a not yet identified medium compound which is followed by switching on a new metabolic pathway. During this lag, the levels of bulk protein are maintained. Our results suggest that a transient drop in the intracellular RNase E level is a means of cells to retard mRNA turnover in a period of adjustment to medium utilization. Furthermore, the here described regulation of the rne transcript and its cognate gene product seems to occur by an RNase E-independent mechanism responsive to changes in growth conditions.

NIPP-1, a nuclear inhibitory subunit of protein phosphatase-1, has RNA-binding properties.

NIPP-1 is a nuclear inhibitory subunit of protein phosphatase-1 with structural similarities to some proteins involved in RNA processing. We report here that baculovirus-expressed recombinant NIPP-1 displays RNA-binding properties, as revealed by North-Western analysis, by UV-mediated cross-linking, by RNA mobility-shift assays, and by chromatography on poly(U)-Sepharose. NIPP-1 preferentially bound to U-rich sequences, including RNA-destabilizing AUUUA motifs. NIPP-1 also associated with single-stranded DNA, but had no affinity for double-stranded DNA. The binding of NIPP-1 to RNA was blocked by antibodies directed against the COOH terminus of NIPP-1, but was not affected by prior phosphorylation of NIPP-1 with protein kinase A or casein kinase-2, which decreases the affinity of NIPP-1 for protein phosphatase-1. The catalytic subunit of protein phosphatase-1 did not bind to poly(U)-Sepharose, but it bound very tightly after complexation with NIPP-1. These data are in agreement with a function of NIPP-1 in targeting protein phosphatase-1 to RNA.

Biochemical and serological evidence for an RNase E-like activity in halophilic Archaea.

Endoribonuclease RNase E appears to control the rate-limiting step that mediates the degradation of many mRNA species in bacteria. In this work, an RNase E-like activity in Archaea is described. An endoribonucleolytic activity from the extreme halophile Haloarcula marismortui showed the same RNA substrate specificity as the Escherichia coli RNase E and cross-reacted with a monoclonal antibody raised against E. coli RNase E. The archaeal RNase E activity was partially purified from the extreme halophilic cells and shown, contrary to the E. coli enzyme, to require a high salt concentration for cleavage specificity and stability. These data indicate that a halophilic RNA processing enzyme can specifically recognize and cleave mRNA from E. coli in an extremely salty environment (3 M KCI). Having recently been shown in mammalian cells (A. Wennborg, B. Sohlberg, D. Angerer, G. Klein, and A. von Gabain, Proc. Natl. Acad. Sci. USA 92:7322-7326, 1995), RNase E-like activity has now been identified in all three evolutionary domains: Archaea, Bacteria, and Eukarya. This strongly suggests that mRNA decay mechanisms are highly conserved despite quite different environmental conditions.

Repression of beta interferon gene expression in virus-infected cells is correlated with a poly(A) tail elongation.

Expression of beta interferon (IFN-beta) is transiently induced when Namalwa B cells (Burkitt lymphoma cell line) are infected by Sendai virus. In this study, we found that an elongation of the IFN-beta mRNA could be detected in virus-infected cells and that such a modification was not observed when the IFN-beta transcript was induced by a nonviral agent, poly(I-C). Treatment of the cells with a transcriptional inhibitor (actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) resulted in further elongation of the transcript. Characterization of the elongated IFN-beta transcript by primer extension and RNase H treatment showed that the modification was a result of an elongated poly(A) tail of up to 400 nucleotides. We conclude that the poly(A) tail elongation of the IFN-beta transcript is associated with the viral infection. Furthermore, the presence of the elongated IFN-beta transcript correlated with a decrease of IFN-beta protein in the medium and in cell extracts. Sucrose gradient analysis of cytoplasmic extracts showed that IFN-beta transcripts with elongated poly(A) tails were found in the nonpolysomal fractions, whereas the shorter transcripts could be detected in both polysomal and nonpolysomal fractions. A longer form of the IFN-beta mRNA was also found in the nonpolysomal fractions of cells not treated with transcriptional inhibitors. Thus, the observed regulation of IFN-beta mRNA is not entirely dependent on the inhibition of transcription. To our knowledge, this study provides the first example of a poly(A) tail elongation in somatic cells that negatively influences gene expression in vivo.