The leishmaniases are protozoal diseases that severely affect large populations in tropical and subtropical regions. There are only limited treatment options and preventative measures. Vaccines will be important for prevention, control and elimination of leishmaniasis, and could reduce the transmission and burden of disease in endemic populations. We report the development of a DNA vaccine against leishmaniasis that induced T cell-based immunity and is a candidate for clinical trials. The vaccine antigens were selected as conserved in various Leishmania species, different endemic regions, and over time. They were tested with T cells from individuals cured of leishmaniasis, and shown to be immunogenic and to induce CD4(+) and CD8(+) T cell responses in genetically diverse human populations of different endemic regions. The vaccine proved protective in a rodent model of infection. Thus, the immunogenicity of candidate vaccine antigens in human populations of endemic regions, as well as proof of principle for induction of specific immune responses and protection against Leishmania infection in mice, provides a viable strategy for T cell vaccine development.
Whereas neutrophil elastase, cathepsin G, and proteinase 3 have been known as granule-associated serine proteases of neutrophils for decades, a fourth member, called neutrophil serine protease 4 (NSP4), was just recently described and provisionally characterized. In this study, we identified NSP4 as a novel azurophil granule protein of neutrophils by Western blot analyses of subcellular fractions as well as by RT-PCR analyses of neutrophil precursors from human bone marrow. The highest mRNA levels were observed in myeloblasts and promyelocytes, similar to myeloperoxidase, a marker of azurophil granules. To determine the extended sequence specificity of recombinant NSP4, we used an iterative fluorescence resonance energy transfer-based optimization strategy. In total, 142 different peptide substrates with arginine in P1 and variations at the P1, P2, P3, P4, and P2 positions were tested. This enabled us to construct an ?1-proteinase inhibitor variant (Ile-Lys-Pro-Arg-/-Ser-Ile-Pro) with high specificity for NSP4. This tailor-made serpin was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils, indicating that NSP4 is fully processed and stored as an already activated enzyme in azurophil granules. Moreover, cathepsin C was identified as the activator of NSP4 in vivo, as cathepsin C deficiency resulted in a complete absence of NSP4 in a Papillon-Lefèvre patient. Our in-depth analysis of NSP4 establishes this arginine-specific protease as a genuine member of preactivated serine proteases stored in azurophil granules of human neutrophils.
Needle-free vaccination, for reasons of safety, economy, and convenience, is a central goal in vaccine development, but it also needs to meet the immunological requirements for efficient induction of prophylactic and therapeutic immune responses. Combining the principles of noninvasive delivery to dendritic cells (DCs) through skin and the immunological principles of cell-mediated immunity, we developed microparticle-based topical vaccines. We show here that the microparticles are efficient carriers for coordinated delivery of the essential vaccine constituents to DCs for cross-presentation of the antigens and stimulation of T-cell responses. When applied to the skin, the microparticles penetrate into hair follicles and target the resident DCs, the immunologically most potent cells and site for induction of efficient immune responses. The microparticle vaccine principle can be applied to different antigen formats such as peptides and proteins, or nucleic acids coding for the antigens.
Dendritic cells (DCs) are key activators of cellular immune responses through their capacity to induce naïve T cells and sustained effector T cell responses. This capacity is a function of their superior efficiency of antigen presentation via MHC class I and class II molecules, and the expression of co-stimulatory cell surface molecules and cytokines. Maturation of DCs is induced by microbial factors via pattern recognition receptors such as Toll-like receptors, pro-inflammatory cytokines or cognate interaction with CD4(+) T cells. Here we show that, unexpectedly, the PanDR helper T cell epitope PADRE, a generic T helper cell antigen presented by a large fraction of HLA-DR alleles, when delivered in particle-bound form induced maturation of human DCs. The DCs that received the particle-bound PADRE displayed all features of fully mature DCs, such as high expression of the co-stimulatory molecules CD80, CD86, CD83, the MHC-II molecule HLA-DR, secretion of high levels of the biologically active IL-12 (IL-12p70) and induction of vigorous proliferation of naïve CD4(+) T cells. Furthermore, the maturation of DCs induced by particle-bound PADRE was shown to involve sphingosine kinase, calcium signaling from internal sources and downstream signaling through the MAP kinase and the p72syk pathways, and finally activation of the transcription factor NF-?B. Based on our findings, we propose that particle-bound PADRE may be used as a DC activator in DC-based vaccines.
Novel nontoxic (S)-2-aminoalkylbenzimidazole derivatives were found to be effective against Candida spp. at low micromolar concentrations using high-throughput screening with infected HeLa cells. A collection of analogues defined the chemical groups relevant for activity. The most active compound was characterized by transcriptional analysis of the response of C. albicans Sc5314. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole had a strong impact on membrane biosynthesis. Testing different clinically relevant pathogenic fungi showed the selectivity of the antimycotic activity against Candida species.
Fungal infections are a serious health problem in clinics, especially in the immune-compromised patient. Disease ranges from widespread superficial infections like vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses, only a limited arsenal of antifungals is available. The most commonly used classes of antifungal compounds used include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapy, significant side effects, and high costs for several antifungals, there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities, we screened a compound library including more than 35,000 individual compounds derived from organic synthesis as well as combinatorial compound collections representing mixtures of compounds for antimycotic activity. In total, more than 100,000 compounds were screened using a new type of activity-selectivity assay, analyzing both the antifungal activity and the compatibility with human cells at the same time. One promising hit, an (S)-2-aminoalkyl benzimidazole derivative, was developed among a series of lead compounds showing potent antifungal activity. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl) benzimidazole showed the highest antifungal activity and the best compatibility with human cells in several cell culture models and against a number of clinical isolates of several species of pathogenic Candida yeasts. Transcriptional profiling indicates that the newly discovered compound is a potential inhibitor of the ergosterol pathway, in contrast to other benzimidazole derivatives, which target microtubules.
Recently, it has been demonstrated that particulate substances penetrate preferentially into the hair follicles and that the penetration depth depends on the particle size. In the present study, the influence of the vehicle of the particulate substances on the penetration depth was investigated. Four different formulations (ethanolic suspension, aqueous suspension, ethanolic gel and aqueous gel) containing peptide-loaded particles of 1 µm in diameter were prepared and applied on porcine ear skin. After penetration, punch biopsies were taken and the penetration depths of the particles were investigated by laser scanning microscopy. The deepest penetration was achieved with the gel formulations demonstrating an influence of the vehicle on the penetration depth of particulate substances.
MHC class II molecules (MHC II) play a pivotal role in the cell-surface presentation of antigens for surveillance by T cells. Antigen loading takes place inside the cell in endosomal compartments and loss of the peptide ligand rapidly leads to the formation of a non-receptive state of the MHC molecule. Non-receptiveness hinders the efficient loading of new antigens onto the empty MHC II. However, the mechanisms driving the formation of the peptide inaccessible state are not well understood. Here, a combined approach of experimental site-directed mutagenesis and computational modeling is used to reveal structural features underlying "non-receptiveness." Molecular dynamics simulations of the human MHC II HLA-DR1 suggest a straightening of the ?-helix of the ?1 domain during the transition from the open to the non-receptive state. The movement is mostly confined to a hinge region conserved in all known MHC molecules. This shift causes a narrowing of the two helices flanking the binding site and results in a closure, which is further stabilized by the formation of a critical hydrogen bond between residues ?Q9 and ?N82. Mutagenesis experiments confirmed that replacement of either one of the two residues by alanine renders the protein highly susceptible. Notably, loading enhancement was also observed when the mutated MHC II molecules were expressed on the surface of fibroblast cells. Altogether, structural features underlying the non-receptive state of empty HLA-DR1 identified by theoretical means and experiments revealed highly conserved residues critically involved in the receptiveness of MHC II. The atomic details of rearrangements of the peptide-binding groove upon peptide loss provide insight into structure and dynamics of empty MHC II molecules and may foster rational approaches to interfere with non-receptiveness. Manipulation of peptide loading efficiency for improved peptide vaccination strategies could be one of the applications profiting from the structural knowledge provided by this study.
Cell-penetrating peptides (CPPs) may have impli-cations in biomedical sciences by improving the delivery of a wide variety of drugs through the membrane barrier. CPPs are generally taken up by endocytotic pathways, and vesicular encapsulation is a limiting factor in the area of intracellular targeting. A novel, cationic cysteine-rich CPP, CyLoP-1, has been developed exhibiting distinguished diffused cytosolic distribution along with endosomal uptake at low micromolar concentrations. Comparative uptake analysis with known CPPs showed CyLoP-1 as a promising delivery vector to access the cytosol in a variety of cell types. In addition to the positively charged residues, the presence of cysteines and tryptophans proved to be essential to maintain its functionality. Also, the oxidation status of the cysteines played an important role for the uptake efficiency of CyLoP-1, with the disulfide-containing form being more effective. The distinct feature of CyLoP-1 to enter the cytosol was further explored by the covalent attachment of cargoes of different nature and sizes. In particular, induction of caspase-3 activity (indicating apoptosis) by a CyLoP-1-SmacN7 conjugate proved successful delivery of the pro-apoptotic cargo to its site of action in the cytosol. Efficient intracellular delivery into the entire cytosol already at low micromolar concentrations makes CyLoP-1 a promising candidate for cytosolic delivery of cargoes of small sizes. Thus, this peptide might prove to be useful for efficient transmembrane delivery of agents directed to cytosolic targets.
The ATP-binding cassette transporter associated with antigen processing (TAP) plays a key role in the adaptive immune defense against infected or malignantly transformed cells by translocating proteasomal degradation products into the lumen of the endoplasmic reticulum for loading onto MHC class I molecules. The broad substrate spectrum of TAP, rendering peptides from 8 to 40 residues, including even branched or modified molecules, suggests an unforeseen structural flexibility of the substrate-binding pocket. Here we used EPR spectroscopy to reveal conformational details of the bound peptides. Side-chain dynamics and environmental polarity were derived from covalently attached 2,2,5,5-tetramethylpyrrolidine-1-oxyl spin probes, whereas 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin-labeled peptides were used to detect backbone properties. Dependent on the spin probes position, striking differences in affinity, dynamics, and polarity were found. The side-chains mobility was strongly restricted at the ends of the peptide, whereas the central region was flexible, suggesting a central peptide bulge. In the end, double electron electron resonance allowed the determination of intrapeptide distances in doubly labeled peptides bound to TAP. Simulations based on a rotamer library led to the conclusion that peptides bind to TAP in an extended kinked structure, analogous to those bound to MHC class I proteins.
T-cell recognition of peptides bound to MHC class II (MHCII) molecules is a central event in cell-mediated adaptive immunity. The current paradigm holds that prebound class II-associated invariant chain peptides (CLIP) and all subsequent antigens maintain a canonical orientation in the MHCII binding groove. Here we provide evidence for MHCII-bound CLIP inversion. NMR spectroscopy demonstrates that the interconversion from the canonical to the inverse alignment is a dynamic process, and X-ray crystallography shows that conserved MHC residues form a hydrogen bond network with the peptide backbone in both orientations. The natural catalyst HLA-DM accelerates peptide reorientation and the exchange of either canonically or inversely bound CLIP against antigenic peptide. Thus, noncanonical MHC-CLIP displays the hallmarks of a structurally and functionally intact antigen-presenting complex.
The identification of epitopes that elicit cytotoxic T-lymphocyte activity is a prerequisite for the development of cancer-specific immunotherapies. However, especially the parallel characterization of several epitopes is limited by the availability of T cells. Microarrays have enabled an unprecedented miniaturization and parallelization in biological assays. Here, we developed peptide microarrays for the detection of CTL activity. MHC class I-binding peptide epitopes were pipetted onto polymer-coated glass slides. Target cells, loaded with the cell-impermeant dye calcein, were incubated on these arrays, followed by incubation with antigen-expanded CTLs. Cytotoxic activity was detected by release of calcein and detachment of target cells. With only 200,000 cells per microarray, CTLs could be detected at a frequency of 0.5% corresponding to 1,000 antigen-specific T cells. Target cells and CTLs only settled on peptide spots enabling a clear separation of individual epitopes. Even though no physical boundaries were present between the individual spots, peptide loading only occurred locally and cytolytic activity was confined to the spots carrying the specific epitope. The peptide microarrays provide a robust platform that implements the whole process from antigen presentation to the detection of CTL activity in a miniaturized format. The method surpasses all established methods in the minimum numbers of cells required. With antigen uptake occurring on the microarray, further applications are foreseen in the testing of antigen precursors that require uptake and processing prior to presentation.
Respiratory syncytial virus (RSV) is an important cause of acute respiratory disease in infants, immunocompromised subjects and the elderly. However, it is unclear why most primary RSV infections are associated with relatively mild symptoms, whereas some result in severe lower respiratory tract infections and bronchiolitis. Since RSV hospitalization has been associated with respiratory bacterial co-infections, we have tested if bacterial Toll-like receptor (TLR) agonists influence RSV-A2-GFP infection in human primary cells or cell lines. The synthetic bacterial lipopeptide Pam3-Cys-Ser-Lys4 (Pam3CSK4), the prototype ligand for the heterodimeric TLR1/TLR2 complex, enhanced RSV infection in primary epithelial, myeloid and lymphoid cells. Surprisingly, enhancement was optimal when lipopeptides and virus were added simultaneously, whereas addition of Pam3CSK4 immediately after infection had no effect. We have identified two structurally related lipopeptides without TLR-signaling capacity that also modulate RSV infection, whereas Pam3CSK4-reminiscent TLR1/2 agonists did not, and conclude that modulation of infection is independent of TLR activation. A similar TLR-independent enhancement of infection could also be demonstrated for wild-type RSV strains, and for HIV-1, measles virus and human metapneumovirus. We show that the effect of Pam3CSK4 is primarily mediated by enhanced binding of RSV to its target cells. The N-palmitoylated cysteine and the cationic lysines were identified as pivotal for enhanced virus binding. Surprisingly, we observed inhibition of RSV infection in immortalized epithelial cell lines, which was shown to be related to interactions between Pam3CSK4 and negatively charged glycosaminoglycans on these cells, which are known targets for binding of laboratory-adapted but not wild-type RSV. These data suggest a potential role for bacterial lipopeptides in enhanced binding of RSV and other viruses to their target cells, thus affecting viral entry or spread independent of TLR signaling. Moreover, our results also suggest a potential application for these synthetic lipopeptides as adjuvants for live-attenuated viral vaccines.
The pattern recognition receptors (PRRs) of the innate immune system are the first defence line of the immune system. Toll-like receptors (TLRs) are the most well known and the best examined of the PR receptors. In the last years TLRs had been studied in different ways resulting in a lot of new insights in the function and signalling pathways of these receptors. However, it was not possible to investigate individual combinations of the TLRs and their specific ligands, because of the complex network in immune signalling resulting in interference with each other. This work shows a new cell-based assay, established for the analysis of single PRRs or heterodimers. For this purpose NIH3T3 (mouse fibroblasts) were stably transfected with the NF-kappaB-inducible reporter gene secreted alkaline phosphatase (SEAP) together with the corresponding combinations of human TLRs and their co-receptors (e.g. TLR1/2, TLR2/6 and TLR4/CD14). The specificity of the respective cell lines was shown by induction with variations of specific and unspecific ligands (immune-stimulating components of microorganisms or synthetic ligands). Analysis via the NF-kappaB-dependent reporter gene SEAP allows a direct way to detect the human TLR-activity. Our results showed that this assay is highly sensitive and specific for the respective ligands. For the synthetic ligands Pam(2)CysSK(4) the assay demonstrates a detection limit of 1 pg/ml for TLR2/6. In summary, this test system allows the investigation of individual human PRR-receptors in a highly specific way, without interference with other immune components opening new avenues for novel insights in the innate immune system and its applications.
Chemical biology aims for a perfect control of protein complexes in time and space by their site-specific labeling, manipulation, and structured organization. Here we developed a self-inactivated, lock-and-key recognition element whose binding to His-tagged proteins can be triggered by light from zero to nanomolar affinity. Activation is achieved by photocleavage of a tethered intramolecular ligand arming a multivalent chelator head for high-affinity protein interaction. We demonstrate site-specific, stable, and reversible binding in solution as well as at interfaces controlled by light with high temporal and spatial resolution. Multiplexed organization of protein complexes is realized by an iterative in situ writing and binding process via laser scanning microscopy. This light-triggered molecular recognition should allow for a spatiotemporal control of protein-protein interactions and cellular processes by light-triggered protein clustering.
Experimental results are presented for 180 in silico designed octapeptide sequences and their stabilizing effects on the major histocompatibility class I molecule H-2K(b). Peptide sequence design was accomplished by a combination of an ant colony optimization algorithm with artificial neural network classifiers. Experimental tests yielded nine H-2K(b) stabilizing and 171 nonstabilizing peptides. 28 among the nonstabilizing octapeptides contain canonical motif residues known to be favorable for MHC I stabilization. For characterization of the area covered by stabilizing and non-stabilizing octapeptides in sequence space, we visualized the distribution of 100,603 octapeptides using a self-organizing map. The experimental results present evidence that the canonical sequence motives of the SYFPEITHI database on their own are insufficient for predicting MHC I protein stabilization.
Toll-like receptors (TLR) are highly conserved pattern recognition receptors of the innate immune system. Toll-like receptor 2 (TLR2) recognizes bacterial lipopeptides in a heterodimeric complex with TLR6 or TLR1, thereby discriminating between di- or triacylated lipopeptides, respectively. Previously, we found that HEK293 cells transfected with bovine TLR2 (boTLR2) were able to respond to diacylated lipopeptides but did not recognize triacylated lipopeptides, even after cotransfection with the so far published sequence of boTLR1. In this study we now could show that primary bovine cells were in general able to detect triacylated lipopetides. A closer investigation of the boTLR1 gene locus revealed an additional ATG 195 base pairs upstream from the published start codon. Its transcription would result in an N-terminus with high identity to human and murine TLR1 (huTLR1, muTLR1). Cloning and cotransfection of this longer boTLR1 with boTLR2 now resulted in the recognition of triacylated lipopeptides by HEK293 cells, thereby resembling the ex vivo observation. Analysis of the structure-activity relationship showed that the ester-bound acid chains of these lipopeptides need to consist of at least 12 carbon atoms to activate the bovine heterodimer showing similarity to the recognition by huTLR2/huTLR1. In contrast, HEK293 cell cotransfected with muTLR2 and muTLR1 could already be activated by lipopeptides with shorter fatty acids of only 6 carbon atoms. Thus, our data indicate that the additional N-terminal nucleotides belong to the full length and functionally active boTLR1 (boTLR1-fl) which participates in a species-specific recognition of bacterial lipopeptides.
The innate immune response provides a critical first-line defense against Mycobacterium tuberculosis, an intracellular pathogen that represents a major health threat world-wide. A synthetic lipopeptide (LP) mimicking the lipid moiety of the cell-wall associated 19-kDa lipoprotein from M. tuberculosis has recently been assigned an important role in the induction of an antibacterial immune response in host macrophages. Here, we present experimental data on the biological activities and the biophysical mechanisms underlying cell activation by synthetic 19-kDa M. tuberculosis-derived lipopeptide (Mtb-LP). Investigation of the geometry of the LP (i.e. the molecular conformation and supramolecular aggregate structure) and the preference for membrane intercalation provide an explanation for the biological activities of the mycobacterial LP. Cell activation by low concentrations of Mtb-LP was enhanced by the lipopolysaccharide-binding protein and CD14. However, surprisingly, we found that activation of human macrophages to induce pro- as well as antiinflammatory mediators (tumor necrosis factor(TNF)-alpha, Interleukin(IL)-6, IL-8, and IL-10) in response to the Mtb-LP is strongly reduced in the presence of serum. This observation could be confirmed for the immune response of murine macrophages which showed a strongly enhanced TNF-alpha release in the absence of serum, suggesting that the molecular mechanisms of immune recognition of the Mtb-LP are tailored to the ambient conditions of the lung.
The development of synthetic, low-molecular-weight ligand receptor systems for the selective control of biomolecular interactions remains a major challenge. Binding of oligohistidine peptides to chelators containing Ni2+-loaded nitrilotriacetic acid (NTA) moieties is one of the most widely used and best-characterised recognition systems. Recognition units containing multiple NTA moieties (multivalent chelator headgroups, MCHs) recognise oligohistidines with substantially increased binding affinities. Different multivalencies both at the level of the MCH and at that of the oligohistidine ligand provide a powerful means to vary the affinity of the interaction systematically. Here we have explored the selectivity for the binding of different oligohistidines to immobilised MCH. Using microarrays of mono-, bis-, tris- and tetrakis-NTA chelators spotted at different surface densities, we explored the ability of these binders to discriminate fluorescently labelled hexa- and decahistidine peptides. When hexa- and decahistidine were tested alone, the discrimination of ligands showed little dependence either on the nature or on the density of the chelator. In contrast, coincubation of both peptides decreased the affinity of hexahistidine, increased the affinity of decahistidine, and made the binding of decahistidine highly dependent on MCH density. Kinetic binding assays by dual-colour total internal reflection fluorescence spectroscopy revealed active exchange of His6 by His10 and confirmed the high selectivity towards His10. Our results establish the key role of surface multivalency for the selectivity of multivalent interactions at interfaces.
Combination therapies have proven vital in the fight against HIV and cancer. However, the identification and optimization of such combination therapies is largely experience driven and an activity of clinicians rather than of systematic screening efforts. Here we present a diffusion device, compatible with the format of a 12-well microtiter plate, to create and test all possible mixtures of two substances with only two pipetting steps. Applications to the testing of different drug combinations and the parallel screening of different leukemia cell lines as well as primary patient cells are presented. The diffusion device yields qualitatively and quantitatively comparable results to an MTT viability assay conducted in a standard 96-well format albeit with a tremendous reduction of processing steps. In addition, a fluorescence-based annexin V binding assay of cell death was implemented. Next to the reduction of processing steps, the diffusion device constitutes a considerable assay miniaturization that overcomes the problems typically associated with miniaturization as a consequence of small sample volumes. Given its ease of handling, the device will greatly advance the development and optimization of combination drugs and the identification of optimum drug combinations in personalized medicine.
Despite intensive research on the identification of T-cell epitopes in cattle after foot-and-mouth disease virus (FMDV) infection during the last 20 years, knowledge of major histocompatibility complex (MHC) restriction and anchor residues of such epitopes is still sparse. Therefore, as a first step, we tested lymphocytes from two experimentally FMDV serotype A24-vaccinated and -challenged cattle for recognition of FMDV-derived pentadecapeptides in proliferation assays. Two epitopes were identified: amino acid residues 66 to 80 within the structural protein 1D and amino acid residues 22 to 36 within the structural protein 1A. The latter epitope was recognized by lymphocytes from both cattle. Peptide-specific proliferation was caused by a response of CD4(+) T helper cells as identified by carboxyfluorescein diacetate succinimidyl ester proliferation assays. Having identified one epitope that was recognized by two cattle, we hypothesized that these animals should have common MHC class II alleles. Cloning and sequencing of DRB3, DQA, and DQB alleles revealed that both animals possessed DQA allele 22021 and DQB allele 1301 but had no common DRB3 allele. A parallel analysis of amino acid residues involved in MHC presentation by peptides with alanine substitutions showed that the amino acid residues in positions 5 and 9 within the pentadecapeptide representing the 1A epitope were important for MHC binding in both cattle. These data indicate that the epitope located on FMDV protein 1A can be presented by MHC class II DQ molecules encoded by DQA allele 22021 and DQB allele 1301 and present the first evidence of the binding motif of this particular DQ molecule.
Regulators of G-protein signalling accelerate the GTPase activity of G(alpha) subunits, driving G proteins in their inactive GDP-bound form. This property defines them as GTPase activating proteins. Here the effect of different Toll-like receptor agonists on RGS1 and RGS2 expression in murine bone marrow-derived macrophages and J774 cells was analysed. After stimulation with TLR2/1 or TLR2/6 lipopeptide ligands and the TLR4/MD2 ligand lipopolysaccharide, microarray analyses show only modulation of RGS1 and RGS2 among all the regulators of G-protein signalling tested. Real-time PCR confirmed modulation of RGS1 and RGS2. In contrast to RGS2, which was always downregulated, RGS1 mRNA was upregulated during the first 30 min after stimulation, followed by downregulation. Similar results were also found in the murine macrophage cell line J774. The ligand for intracellular TLR9 modulates RGS1 and RGS2 in a similar manner. However, the TLR3 ligand poly(I:C) permanently upregulates RGS1 and RGS2 expression indicating a different modulation by the MyD88- and TRIF-signalling pathway. This was confirmed using MyD88(-/-) and TRIF(-/-) bone marrow-derived macrophages. Modulation of RGS1 and RGS2 by Toll-like receptor ligands plays an important role during inflammatory and immunological reactions after bacterial and viral infection.
Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.
Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. The immune mechanisms, which control viral multiplication and dissemination, are not yet known. Here, we studied the antibody response against the CHIKV surface antigens in infected patients. With plasma samples obtained during the early convalescent phase, we showed that the naturally-acquired IgG response is dominated by IgG3 antibodies specific mostly for a single linear epitope E2EP3. E2EP3 is located at the N-terminus of the E2 glycoprotein and prominently exposed on the viral envelope. E2EP3-specific antibodies are neutralizing and their removal from the plasma reduced the CHIKV-specific antibody titer by up to 80%. Screening of E2EP3 across different patient cohorts and in non-human primates demonstrated the value of this epitope as a good serology detection marker for CHIKV infection already at an early stage. Mice vaccinated by E2EP3 peptides were protected against CHIKV with reduced viremia and joint inflammation, providing a pre-clinical basis for the design of effective vaccine against arthralgia-inducing CHIKV and other alphaviruses.
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