Release Of Polypeptides Research Articles

Probing the Biosafety of Implantable Artificial Secretory Granules for the Sustained Release of Bioactive Proteins.

Among bio-inspired protein materials, secretory protein microparticles are of clinical interest as self-contained, slow protein delivery platforms that mimic secretory granules of the human endocrine system, in which the protein is both the drug and the scaffold. Upon subcutaneous injection, their progressive disintegration results in the sustained release of the building block polypeptides, which reach the bloodstream for systemic distribution and subsequent biological effects. Such entities are easily fabricated in vitro by Zn-assisted cross-molecular coordination of histidine residues. Using cationic Zn for the assembly of selected pure protein species and in the absence of any heterologous holding material, these granules are expected to be nontoxic and therefore adequate for different clinical uses. However, such presumed biosafety has not been so far confirmed and the potential protein dosage threshold not probed yet. By selecting the receptor binding domain (RBD) from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein as a model protein and using a mouse lab model, we have explored the toxicity of RBD-made secretory granules at increasing doses up to ∼100 mg/kg of animal weight. By monitoring body weight and biochemical blood markers and through the histological scrutiny of main tissues and organs, we have not observed systemic toxicity. Otherwise, the bioavailability of the material was demonstrated by the induction of specific antibody responses. The presented data confirm the intrinsic biosafety of artificial secretory granules made by recombinant proteins and prompt their further clinical development as self-contained and dynamic protein reservoirs.

Bioactive fibrous scaffolds with programmable release of polypeptides regulate inflammation and extracellular matrix remodeling.

Inflammation manipulation and extracellular matrix (ECM) remodeling for healthy tissue regeneration are critical requirements for tissue engineering scaffolds. To this end, the bioactive polycaprolactone (PCL)-based scaffolds are fabricated to release aprotinin and thymosin β4 (Tβ4) in a programmable manner. The core part of the fiber is composed of hyaluronic acid and Tβ4, and the shell is PCL, which is further coated with heparin/gelatin/aprotinin to enhance biocompatibility. The in vitro assay demonstrates that the controlled release of aprotinin prevents initial excessive inflammation. The subsequent release of Tβ4 after 3 days induces the transition of macrophages from M1 into M2 polarization. The manipulation of inflammatory response further controls the expression of transforming growth factor-β and fibroblast activation, which oversee the quantity and quality of ECM remodeling. In addition, the gradual degradation of the scaffold allows cells to proliferate within the platform. In vivo implant evaluation convinces that PCL-based scaffolds possess the high capability to control the inflammatory response and restore the ECM to normal conditions. Hence, our work paves a new way to develop tissue engineering scaffolds for inflammation manipulation and ECM remodeling with peptide-mediated reactions.

Mechanisms of deformation and drug release of targeting polypeptides based on fibronectin induction

Polypeptide nano-carriers with deformation and sustained-release function have gained an attention in anti-tumor treatment. A multifunctional polypeptide with different motifs was discussed and the contribution of each motif to targeted drug release was analyzed by control studies. The transformation and drug release processes of polypeptides were investigated by molecular dynamics method to reveal their dynamics mechanism, and corresponding experiments were performed to verify the simulation results. We observed that the polypeptides could form NPs under the hydrophobic interaction between self-assembly motifs and the electrostatic repulsion between targeting motifs. Affected by the ligand-receptor interaction, the targeting motifs overcame the electrostatic repulsion to approach the ligand proteins, leading to the promotion of the binding of fibrous motifs and the transformation of NPs into NFs for better retention of drugs in the tumor tissues. In addition, the polypeptides with strong hydrophobicity exhibited excellent sustained-release efficiency. These insights allow drawing general conclusions contributed to the design of transformable polypeptide NPs: The decrease in the hydrophobicity of self-assembly motifs is beneficial for the enrichment of doxorubicin in tumor tissues, as well as the similar result can be obtained with the improvement of the hydrophobicity of fibrous motifs and the capability of target.

Membrane-dependent relief of translation elongationarrest on pseudouridine- and N1-methyl-pseudouridine-modified mRNAs.

Expression of therapeutically important proteins has benefited dramatically from the advent of chemically modified mRNAs that feature decreased lability and immunogenicity. This had a momentous effect on the rapid development of COVID-19 mRNA vaccines. Incorporation of the naturally occurring pseudouridine (Ψ) or N1-methyl-pseudouridine (N1mΨ) into in vitro transcribed mRNAs prevents the activation of unwanted immune responses by blocking eIF2α phosphorylation, which inhibits translation. Here, we report that Ψs in luciferase (Luc) mRNA exacerbate translation pausing in nuclease-untreated rabbit reticulocyte lysate (uRRL) and promote the formation of high-order-ribosome structures. The major deceleration of elongation occurs at the Ψ-rich nucleotides 1294–1326 of Ψ-Luc mRNA and results in premature termination of translation. The impairment of translation is mainly due to the shortage of membranous components. Supplementing uRRL with canine microsomal membranes (CMMs) relaxes the impediments to ribosome movement, resolves collided ribosomes, and greatly enhances full-size luciferase production. CMMs also strongly stimulated an extremely inefficient translation of N1mΨ-Luc mRNA in uRRL. Evidence is presented that translational pausing can promote membrane recruitment of polysomes with nascent polypeptides that lack a signal sequence. Our results highlight an underappreciated role of membrane binding to polysomes in the prevention of ribosome collision and premature release of nascent polypeptides.

Conformational equilibria in allosteric control of Hsp70 chaperones

Heat-shock proteins of 70kDa (Hsp70s) are vital for all life and are notably important in protein folding. Hsp70s use ATP binding and hydrolysis at a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides at a substrate-binding domain (SBD); however, the mechanistic basis for this allostery has been elusive. Here, we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp70 DnaK. We then develop a theoretical model for allosteric equilibria among Hsp70 conformational states to explain the observations: a restraining state, Hsp70R-ATP, restricts ATP hydrolysis and binds peptides poorly, whereas a stimulating state, Hsp70S-ATP, hydrolyzes ATP rapidly and has high intrinsic substrate affinity but rapid binding kinetics. We support this model for allosteric regulation with DnaK structures obtained in the postulated stimulating state S with biochemical tests of the S-state interface and with improved peptide-binding-site definition in an R-state structure.

PBAT hollow porous microfibers prepared via electrospinning and their functionalization for potential peptide release

Although microfiber (MF) loaded polypeptides have been widely studied in the field of medicine, load control and slow release remain significant challenges. Herein, polybutylene adipate terephthalate (PBAT) hollow porous MFs (HPMFs) were prepared by improving and regulating the groups and structures of the MFs to address these challenges. The capacity of the HPMFs for the loading of polypeptide can be improved. Measurements involving the use of X-ray photoelectron, energy-dispersive, and ultraviolet (UV) spectroscopies in conjunction with scanning electron microscopy measurements showed that the amount of polypeptide (arginylglycylaspartic acid, RGD) loaded on the HPMFs was significantly higher than that loaded on the MFs. Animal cell experiments revealed that PBAT grafted MAH has good bioactivity. By loading RGD onto HPMFs and MFs, RGD@HPMFs and RGD@MFs were obtained. A study on HeLa and A549 cancer cells showed that the inhibition rates of RGD@HPMFs were higher than that of RGD@MFs by 14.1% and 6.9%, respectively. The results obtained herein show that HPMF scaffold preparation by improving the material groups and regulating the structure of MFs can address the challenges associated with control of the load and sustained release of polypeptides and other drugs.

Theory of Allosteric Regulation in Hsp70 Molecular Chaperones.

Heat-shock proteins of 70 kDa (Hsp70s) are ubiquitous molecular chaperones that function in protein folding as well as other vital cellular processes. They bind and hydrolyze ATP in a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides in a substrate-binding domain (SBD). However, the molecular mechanism for this allosteric action has remained unclear. Here, we develop and experimentally quantify a theoretical model for Hsp70 allostery based on equilibria among Hsp70 conformational states. We postulate that, when bound to ATP, Hsp70 is in equilibrium between a restraining state (R) that restricts ATP hydrolysis and binds peptides poorly, if at all, and a stimulating state (S) that hydrolyzes ATP relatively rapidly and has high intrinsic substrate affinity but rapid binding kinetics; after the hydrolysis to ADP, NBD and SBD disengage into an uncoupled state (U) that binds peptide substrates tightly, but now with slow kinetics of exchange.

Cheese proteolysis and matrix disintegration during in vitro digestion

Abstract The proteolytic changes under in vitro digestive conditions were examined by urea-polyacrylamide gel electrophoresis, Kjeldahl determination of nitrogen, and fluorescamine analysis to evaluate the level of free amino terminal groups as an indicator of the degree of proteolysis. The protein and fat components of cheese digesta were visualized by confocal laser scanning microscopy. Cheese disintegration and subsequent release of proteins and polypeptides was quantified after each of three in vitro digestion phases: oral, gastric (pepsin, pH 2), and intestinal (pancreatic enzymes, pH 6.8). Most of the disintegration of the cheese protein matrix (˜75%), proteolysis, and protein release occurred during the intestinal digestion phase. Fat aggregate release from the cheese matrix mostly occurred in the gastric and intestinal digestion phases, and globules became more spherical after the gastric phase. Proteolysis took place primarily in the gastric and intestinal phases. These results show the importance of microstructure and the digestive environment on the release of cheese components.

Thermally-Responsive Loading and Release of Elastin-Like Polypeptides from Contact Lenses.

Contact lenses are widely prescribed for vision correction, and as such they are an attractive platform for drug delivery to the anterior segment of the eye. This manuscript explores a novel strategy to drive the reversible adsorption of peptide-based therapeutics using commercially available contact lenses. To accomplish this, thermo-sensitive elastin-like polypeptides (ELPs) alone or tagged with a candidate ocular therapeutic were characterized. For the first time, this manuscript demonstrates that Proclear CompatiblesTM contact lenses are a suitable platform for ELP adsorption. Two rhodamine-labelled ELPs, V96 (thermo-sensitive) and S96 (thermo-insensitive), were employed to test temperature-dependent association to the contact lenses. During long-term release into solution, ELP coacervation significantly modulated the release profile whereby more than 80% of loaded V96 retained with a terminal half-life of ~4 months, which was only 1–4 days under solubilizing conditions. A selected ocular therapeutic candidate lacritin-V96 fusion (LV96), either free or lens-bound LV96, was successfully transferred to HCE-T cells. These data suggest that ELPs may be useful to control loading or release from certain formulations of contact lenses and present a potential for this platform to deliver a biologically active peptide to the ocular surface via contact lenses.

Inter-polysomal coupling of termination and initiation during translation in eukaryotic cell-free system.

The recording of the luciferase-generated luminescence in the eukaryotic cell-free translation system programmed with mRNA encoding firefly luciferase (Luc-mRNA) showed that the addition of free exogenous mRNAs into the translation reactor induces the immediate release of the functionally active protein from the polyribosomes of the translation system. The phenomenon did not depend on the coding specificity of the added free mRNA. At the same time it depended on the “initiation potential” of the added mRNA (including the features that ensure the successful initiation of translation, such as the presence of the cap structure and the sufficient concentration of the added mRNA in the translation mixture). The phenomenon also strictly depended on the presence of the stop codon in the translated mRNA. As the above-mentioned features of the added mRNA imply its activity in initiation of a new translation, the experimental data are found in agreement with the scenario where the molecules of the added mRNA interact by their 5′-ends with terminating and recycling ribosomes, stimulating the release of the complete polypeptides and providing for the initiation of a new translation.

Mechanism of interaction of Al3+ with the proteins composition of photosystem II.

The inhibitory effect of Al3+on photosystem II (PSII) electron transport was investigated using several biophysical and biochemical techniques such as oxygen evolution, chlorophyll fluorescence induction and emission, SDS-polyacrylamide and native green gel electrophoresis, and FTIR spectroscopy. In order to understand the mechanism of its inhibitory action, we have analyzed the interaction of this toxic cation with proteins subunits of PSII submembrane fractions isolated from spinach. Our results show that Al 3+, especially above 3 mM, strongly inhibits oxygen evolution and affects the advancement of the S states of the Mn4O5Ca cluster. This inhibition was due to the release of the extrinsic polypeptides and the disorganization of the Mn4O5Ca cluster associated with the oxygen evolving complex (OEC) of PSII. This fact was accompanied by a significant decline of maximum quantum yield of PSII (Fv/Fm) together with a strong damping of the chlorophyll a fluorescence induction. The energy transfer from light harvesting antenna to reaction centers of PSII was impaired following the alteration of the light harvesting complex of photosystem II (LHCII). The latter result was revealed by the drop of chlorophyll fluorescence emission spectra at low temperature (77 K), increase of F0 and confirmed by the native green gel electrophoresis. FTIR measurements indicated that the interaction of Al 3+ with the intrinsic and extrinsic polypeptides of PSII induces major alterations of the protein secondary structure leading to conformational changes. This was reflected by a major reduction of α-helix with an increase of β-sheet and random coil structures in Al 3+-PSII complexes. These structural changes are closely related with the functional alteration of PSII activity revealed by the inhibition of the electron transport chain of PSII.

Release factor eRF3 mediates premature translation termination on polylysine-stalled ribosomes in Saccharomyces cerevisiae.

Ribosome stalling is an important incident enabling the cellular quality control machinery to detect aberrant mRNA. Saccharomyces cerevisiae Hbs1-Dom34 and Ski7 are homologs of the canonical release factor eRF3-eRF1, which recognize stalled ribosomes, promote ribosome release, and induce the decay of aberrant mRNA. Polyadenylated nonstop mRNA encodes aberrant proteins containing C-terminal polylysine segments which cause ribosome stalling due to electrostatic interaction with the ribosomal exit tunnel. Here we describe a novel mechanism, termed premature translation termination, which releases C-terminally truncated translation products from ribosomes stalled on polylysine segments. Premature termination during polylysine synthesis was abolished when ribosome stalling was prevented due to the absence of the ribosomal protein Asc1. In contrast, premature termination was enhanced, when the general rate of translation elongation was lowered. The unconventional termination event was independent of Hbs1-Dom34 and Ski7, but it was dependent on eRF3. Moreover, premature termination during polylysine synthesis was strongly increased in the absence of the ribosome-bound chaperones ribosome-associated complex (RAC) and Ssb (Ssb1 and Ssb2). On the basis of the data, we suggest a model in which eRF3-eRF1 can catalyze the release of nascent polypeptides even though the ribosomal A-site contains a sense codon when the rate of translation is abnormally low.

Regulating the degradation rate of silk fibroin films through changing the genipin crosslinking degree

Degradation behavior is a key fundamental topic in the field of silk-based biomaterials. In this study, genipin-crosslinked silk fibroin films with varying crosslinking degrees were generated to investigate the effect of the crosslinking degree on the degradation behavior. Higher crosslinking degrees resulted in increased inter-intramolecular network crosslinking density through covalent bonds, which restricted enzymatic attacks and the release of enzyme-degraded polypeptides from silk fibroin molecular networks, providing greater resistance to enzyme degradation. Furthermore, genipin crosslinking induced the conformational transition from random coil to β-sheet due to the structural rearrangement of the chains to form covalent bonds. High β-sheet content contributes to a more crystalline structure that also enhances resistance to enzyme degradation. Consequently, changing the crosslinking degree had a substantial impact on the degradation rate. The high-crosslinking films (with a crosslinking degree greater than 90%) showed a similar degradability to 75% ethanol-treated films, which were barely degraded by collagenase IA and subcutaneous implantation in SD rats. However, decreasing the crosslinking degree from 90% to 78% significantly increased the degradation ratio from 4 wt% to 18 wt% after 28 days in vivo degradation, respectively. In particular, the degradability was strongly correlated to the crosslinking degree in the low-crosslinking (lower than 90%) films. These results reveal that the crosslinking process can efficiently control molecular structures and regulate the crystallization of silk fibroin materials, in turn providing control of the degradation rate.

The effect of Cd2+ on the release of proteins from thylakoid membranes of tomato leaves

The polypeptide pattern of the thylakoid membrane was investigated to elucidate the decrease of photosystem II (PSII) activity due to Cd<sup>2+</sup> incubation. The release of polypeptides depended on the concentration of the metal and time of incubation. The complete release of the 33 kDa polypeptide and partial release of 18, 23 kDa and I atom of Mn/400 chlorophyll indicated some disorganization of the water splitting system. Partial removal of numerous polypeptides, especially that of the PSII complex and coupling factor (CF<sub>O</sub>, CF<sub>1</sub>), was additionally observed. The Cd<sup>2+</sup>-induced release of thylakoid membrane proteins was not only limited to the PSII donor side, however the latter was particularly sensitive to Cd<sup>2+</sup>.

Self-Assembly of VPS41 Promotes Sorting Required for Biogenesis of the Regulated Secretory Pathway

The regulated release of polypeptides has a central role in physiology, behavior, and development, but the mechanisms responsible for production of the large dense core vesicles (LDCVs) capable of regulated release have remained poorly understood. Recent work has implicated cytosolic adaptor protein AP-3 in the recruitment of LDCV membrane proteins that confer regulated release. However, AP-3 inmammals has been considered to function in theendolysosomal pathway and in the biosynthetic pathway only in yeast. We now find that the mammalian homolog of yeast VPS41, a member of the homotypic fusion and vacuole protein sorting (HOPS) complex that delivers biosynthetic cargo to the endocytic pathway in yeast, promotes LDCV formation through a common mechanism with AP-3, indicating a conserved role for these proteins in the biosynthetic pathway. VPS41 also self-assembles into a lattice, suggesting that it acts as a coat protein for AP-3 in formation of the regulated secretory pathway.

Impact of sourdough on buckwheat flour, batter and bread: Biochemical, rheological and textural insights

In this study, the impact of sourdough fermentation on the biochemical, rheological and bread-making performances of buckwheat flour was investigated. In order to assess the effects of the solely acidification, a chemically acidified batter with the same pH of the sourdough was prepared. Extensive hydrolysis of the globulin fraction and release of small polypeptides occurred upon fermentation. A major reduction in the extent of interactions was observed in the sourdough, mainly due to the modification of the major structural components of buckwheat during the fermentation. The hydrolysis of proteins and, possibly, starch was in turn responsible for the major decrease in elasticity observed in the sourdough. In the batters destined for bread-making, the presence of acids was the major cause for reduced elasticity and increased strength, which could be related to the enhanced water-holding capacity of the proteins and/or protein/starch complexes. The addition of sourdough induced dramatic inhibition of the CO 2 production by the baker’s yeasts during proofing, resulting in lower volume and harder crumb of the sourdough bread. On the other hand, the solely acidification induced hardening of the starch gel upon cooking, which was responsible for lower volume and irregular crumb grain in buckwheat bread.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca<sup>2+</sup> Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca<sup>2+</sup> Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Proteolysis at the plasma membrane of tobacco roots: Biochemical evidence and possible roles

Plasma membrane-associated proteases (pm-proteases) exist principally in roots of Nicotiana tabacum cv. Samsun, whereas in plasma membrane (pm) vesicles prepared from leaves, protease activity was at the detection limit. Biochemical characterisation revealed a high diversity of particular hydrophobic pm-proteases indicating multiple functions in root tissue. One proportion of chromatographically separated proteases was split up by non-reducing SDS-PAGE in 8-12 single polypeptides, dependent on plant nitrogen nutrition. The active polypeptides could be grouped in those that were (i) inhibited, (ii) stimulated and (iii) independent of bivalent cations. Although, the total specific protease activity of various pm vesicles was almost identical, the composition and activity of individual polypeptides was dependent on nitrogen supply of the plants. Particularly, nitrogen deficiency stimulated the activity of high molecular mass proteases (125 kDa-97 kDa), whereas sufficient nitrate supply enhanced proteolytic activity of 90 kDa, 83 kDa and 65 kDa polypeptides. Endogenous proteolysis within pm vesicles suggested that at least partly protease substrates are localised within the same membrane. A comparison of polypeptides originated from proteolysis of pm vesicles and those exudated by roots into the external medium points to a role of root pm-proteases in the specific release of polypeptides into the rhizosphere.

Development of a tripartite vector system for live oral immunization using a Gram-negative probiotic carrier

The mucosa represents the primary target site and thus the first barrier for most microbial pathogens. Nevertheless, nearly all present-day vaccines are applied by an invasive route, target the systemic immune system, and do not confer efficient mucosal protection. Currently, mucosal immunity can only be achieved by the delivery of antigens via the mucosal route. Therefore, multiple efforts are under way to develop mucosal vaccines and particularly live oral vaccines as these would confer considerable advantages. We have engineered the AIDA autotransporter system for the surface presentation and/or release of heterologous polypeptides. This study is focused on the development and evaluation of a tripartite live bacterial vector system for oral vaccination based on the AIDA autotransporter, heterologous virulence factor-derived (poly-)peptides, and the apathogenic Escherichia coli Nissle 1917 strain as a live carrier. Potentially with this system also attenuated Salmonella or Shigella strains might be employed as carriers. Model antigens included e.g. the p60 antigen of Listeria monocytogenes, the OspA/OspG antigens of B. burgdorferi, the LT-B subunit of E. coli, and Stx-B subunits of enterohemorrhaghic E. coli (EHEC), all representing crucial virulence factors of important bacterial pathogens. Exemplary oral immunization studies were conducted using different regimes in BALB/c mice with candidate vaccines expressing Stx B-subunits and OspA and OspG proteins. To monitor the induction of immune responses, specific antibody titers in serum as well as secreted mucosal antibodies of local and distal mucosal surfaces were determined. Antigen-specific mucosal as well as systemic antibodies could be induced; however, thus far the response turned out to be heterogeneous and appeared not to be sufficient to mediate protection.