Thomas Bächi

Monoclonal antibodies localize events in the folding, assembly, and intracellular transport of the influenza virus hemagglutinin glycoprotein

We used monoclonal antibodies that recognize monomeric and/or trimeric forms of the influenza virus hemagglutinin (HA) to study biosynthesis of this integral membrane protein in influenza virus-infected cells. We find the following: First, the globular head of the HA folds into its mature conformation in the endoplasmic reticulum prior to the assembly of HA monomers into trimers. Second, trimerization begins within 1 to 2 min following synthesis, with a half-time of approximately 5 min. Third, trimerization occurs only after the HA has been transported from the endoplasmic reticulum. Fourth, newly formed trimers are sensitive to acid-induced conformational alterations associated with viral fusion activity.

A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses

Virus like particles (VLPs) are known to induce potent B cell responses in the absence of adjuvants. Moreover, epitope-specific antibody responses may be induced by VLPs that contain peptides inserted in their immunodominant regions. However, due to steric problems, the size of the peptides capable of being incorporated into VLPs while still permitting capsid assembly, is rather limited. While peptides genetically fused to either the N- or C-terminus of VLPs present fewer assembly problems, the immune responses obtained against such epitopes are often limited, most likely because the epitopes are not optimally exposed. In addition, such particles may be less stable in vivo. Here, we show that peptides and proteins engineered to contain a free cys can be chemically coupled to VLPs formed from the hepatitis B core antigen (HBcAg) containing a lys in the immuno-dominant region. By using this approach steric hindrance of capsid assembly is abrogated. Peptides or protein coupled to VLPs in an oriented fashion are shown to induce strong and protective B cell responses even against self-epitopes in the absence of adjuvants. This molecular assembly system may be used to induce strong B cell responses against most antigens.

Critical Role for Activation of Antigen-Presenting Cells in Priming of Cytotoxic T Cell Responses After Vaccination with Virus-Like Particles

Virus-like particles (VLPs) are known to induce strong Ab responses in the absence of adjuvants. In addition, VLPs are able to prime CTL responses in vivo. To study the efficiency of this latter process, we fused peptide p33 derived from lymphocytic choriomeningitis virus to the hepatitis B core Ag, which spontaneously assembles into VLPs (p33-VLPs). These p33-VLPs were efficiently processed in vitro and in vivo for MHC class I presentation. Nevertheless, p33-VLPs induced weak CTL responses that failed to mediate effective protection from viral challenge. However, if APCs were activated concomitantly in vivo using either anti-CD40 Abs or CpG oligonucleotides, the CTL responses induced were fully protective against infection with lymphocytic choriomeningitis virus or recombinant vaccinia virus. Moreover, these CTL responses were comparable to responses generally induced by live vaccines, because they could be measured in primary ex vivo 51Cr release assays. Thus, while VLPs alone are inefficient at inducing CTL responses, they become very powerful vaccines if applied together with substances that activate APCs.

Cross-presentation of virus-like particles by skin-derived CD8– dendritic cells: a dispensable role for TAP

Virus‐like particles (VLP) induce efficient CTL responses although they do not carry any genetic information. Here, we analyzed MHC class I associated presentation of VLP‐derived CTL‐epitopes in vivo. After intradermal injection of VLP containing the immunodominant epitope (p33) of lymphocytic choriomeningitis virus (p33‐VLP), presentation of peptide p33 in draining lymph nodes was largely restricted to CD8– skin‐derived dendritic cells (DC). Surprisingly, and in contrast to findings with tumor cells, TAP1‐deficient DC and macrophages mediated efficient cross‐presentation of VLP‐derived p33 in vivo and in vitro. However, the ability of TAP1‐deficient DC to cross‐present p33‐VLP was reduced compared to wild‐type DC, indicating that in DC, both TAP‐dependent and TAP‐independent pathways were operative. In contrast, macrophages cross‐presented p33‐VLP normally in the absence of TAP. The TAP‐dependent pathway of cross‐presentation is therefore confined to DC while both macrophages and DC harbor the TAP‐independent pathway. In summary, the results show that VLP‐derived epitopes are cross‐presented by CD8– DC in vivo in a partial TAP‐independent fashion and highlight important differences in the processing machinery of DC versus macrophages.

Cell-surface display of heterologous epitopes on Staphylococcus xylosus as a potential delivery system for oral vaccination

A system has been developed for the surface expression of heterologous receptors on the cell surface of Staphylococcus xylosus. Gene fragments encoding peptides to be displayed on the cell surface can be assembled by applying a polymerization strategy based on the class-IIS restriction enzyme BspMI and thereafter subcloned into an Escherichia coli-staphylococci shuttle vector designed for targeting of produced fusion proteins to the outer cell surface of the Gram-positive host cell. A heterologous receptor was genetically assembled and expressed on the surface of S. xylosus where the separate regions could be independently probed in immunogold assays, using antisera reacting with different regions of the recombinant receptor. In addition, a receptor-specific humoral immune response could be elicited in mice by oral immunization with recombinant S. xylosus cells, suggesting that these type of Gram-positive bacteria might offer potential vehicles for oral vaccination.

Subcellular distribution of S100 proteins in tumor cells and their relocation in response to calcium activation

Abstract S100 proteins, a subgroup of the EF-hand Ca2+-binding protein family, regulate a variety of cellular processes via interaction with different target proteins. Several pathological disorders, including cancer, are linked to altered Ca2+ homeostasis and might involve the multifunctional S100 proteins, which are expressed in a cell- and tissue-specific manner. The present work demonstrates a distinct intracellular localization of S100A6, S100A4, and S100A2 in two tumor cell lines derived from metastatic epithelial breast adenocarcinoma (MDA-MB231) and cervical carcinoma (HeLa). Treatment of the cells by thapsigargin, the ionophore A23187, or cyclic ADP-ribose, to increase [Ca2+]i via different pathways, led to relocation of S100A6 and S100A4 but only partially of the nuclear S100A2, as demonstrated by confocal laser scanning microscopy. These findings support the hypothesis that S100 proteins could play a crucial role in the regulation of Ca2+ homeostasis in cancer cells.

Intramembrane structural differentiation in sendai virus maturation

Abstract Freeze-fracture electron microscopy of plasma membranes of cells infected with Sendai virus reveals patches of virus-specific membrane particles arranged in a crystalline array. These structures are associated with the inner lipid leaflet of the host membrane and develop at a stage following the insertion of the external glycoproteins (spikes) into the cell surface. A crystalline organization of the membrane is recognized in those parts of the cell surface to which the spikes and nucleoprotein strands are recruited for the assembly of viral particles. After the process of viral budding, this type of structural modification of the host membrane persists only for a short time in the viral envelope: newly formed virions possess a crystalline arrangement of membrane particles which is not found in senescent virus particles.

TAP-independent delivery of antigenic peptides to the endoplasmic reticulum: therapeutic potential and insights into TAP-dependent antigen processing.

We have taken several approaches to investigate the capacity of the secretory pathway to liberate major histocompatibility complex (MHC) class I-restricted antigenic peptides from precursor porypeptides. Cells lacking the peptide transporter (TAP) are unable to deliver peptides from cytosolic antigens to class I molecules. TAP can be bypassed by targeting peptides directly to the endoplasmic reticulum (ER) using NH2-terminal signal sequences. This results in the generation of enormous numbers of MHC class I complexes (50,000 peptides/cell), and recombinant vaccinia viruses expressing such peptides are highly immunogenic. In contrast to signal sequence-targeted peptides, peptides are liberated very inefficiently from internal locations in ER-targeted full-length proteins, indicating that the secretory pathway has a limited capacity for generating antigenic peptides from most polypeptide contexts. We have, however, identified a location in proteins from which peptides can be liberated in numerous contexts in the secretory pathway. Placing a number of different peptides at the COOH termini of a secreted protein and two proteins with type II membrane anchors resulted in their TAP-independent presentation. These findings demonstrate that the secretory compartment possesses proteases able to liberate COOH-terminal antigenic peptides from virtually any context, entirely consistent with a role for these proteases in the processing of TAP-transported antigenic peptide precursors.

Surface display on staphylococci: a comparative study

Two different host‐vector expression systems, designed for cell surface display of heterologous receptors on Staphylococcus xylosus and Staphylococcus carnosus, respectively, were compared for the surface display of four variants of a 101 amino acid region derived from the G glycoprotein of human respiratory syncytial virus (RSV). Surface localization of the different chimeric receptors was evaluated by a colorimetric assay and by fluorescence‐activated cell sorting. It was concluded that the S. carnosus system was better both in the ability to translocate inefficiently secreted peptides and in the number of exposed hybrid receptors. The potential use of the described staphylococci as live bacterial vaccine vehicles or alternatives to filamentous phages for surface display of protein libraries is discussed.

Glycophorin-enriched vesicles obtained by a selective extraction of human erythrocyte membranes with a non-ionic detergent

A method is described for isolating glycophorin-enriched vesicles from human erythrocytes by extracting membranes that were incubated for 30 min at 37 degrees C at pH 4.5 and washed at low and high ionic strength with the nonionic detergent Triton X-100. The extracts were 11.8 +/- 2.4 fold enriched in glycophorin and contained 325 +/- 69 microgram sialic acid/mg protein, which represented 61 +/- 16% of the total sialic acid. Upon removal of Triton X-100 one third of the total glycophorin forms glycophorin-enriched vesicles with coextracted, endogenous lipids as shown sedimintation, dextran-density gradient centrifugation, and electron microscopy. Addition of exogenous lipids increased the fraction of glycophorin-enriched vesicles up to 87%. The incorporation of glycophorin in the membrane was shown by hemagglutination inhibition assays using anti-M sera and by the accessibility of glycophorin to trypsin. Freeze-fractured vesicles did not reveal intramembranous particles. The selectivity of the extraction procedure is not simply due to chemical constraints introduced by disulfide cross-linkage of protein component 3, because only 20% of this protein undergo disulfide cross-linking. The selective extraction of glycophorin implies that glycophorin is segregated from protein component 3 and thus from intramembranous particles when erythrocyte membranes have been incubated at pH 4.5. This segregation may precede aggregation of intramembranous particles.