The biological activity of many proteins is influenced by glycosylation, underscoring the essential role of the glycosylation process. However, no established recombinant technique currently enables the controlled shedding of glycosylated extracellular loops from transmembrane proteins. Here, we describe enzymatically controlled release of proteins and peptides (ENCOREP), a strategy that enables in situ expression at the cell surface and protease-mediated release, followed by collection from the cell culture medium. Using ENCOREP, we achieved the production of glycosylated peptides. These glycopeptides, derived from the large extracellular loop of the highly glycosylated CD63, illustrate the type of targets that are otherwise inaccessible with current methods but can be readily obtained using ENCOREP. Overall, ENCOREP provides a rapid and reliable approach to obtain glycosylated proteins or peptides while bypassing the conventional signal peptide-dependent secretory pathway.

Background HIV and HCV share similar routes of infection. Individuals carrying both viruses experience a faster progression of the liver disease. We have analyzed people with new HIV-1 diagnoses in Germany for active or resolved HCV infection for over ten years. The time period covers the introduction of direct acting antivirals (DAA), a paradigmatic shift in HCV therapy. Methods A central component of the HIV surveillance in Germany is the notification of new diagnoses. Residual blood samples from 16,539 people with new HIV-1 diagnoses reported between 2009 and 2019 were examined for HCV antigen and/or antibodies. Reactive cases were further investigated for active or resolved HCV infection by RT-qPCR. The results were analyzed with socio-demographic information from the notification forms. Results The study includes samples from 48.0% of all notified HIV-1 diagnoses. The seroprevalence of cases with HCV antigen, antibodies or both, representing active and resolved infections was 6.2% with stable seroprevalence. The average proportion of resolved infections among those was 33.0% with a significant increase since the introduction of DAAs in 2012 (p Trend_2012-2019 = 0.028) reaching 48.2% in 2019. The highest proportion of active and resolved cases (73.7%) was found in people who inject drugs (PWIDs). This transmission group had the lowest percentage of resolved infections with 29.4%. The proportion of active and resolved cases in persons with heterosexual mode of transmission (HET) and in men who have sex with men (MSM) was 3.8% and 2.6%, respectively. The peak percentage of resolved infections was found in MSM (40.0%), followed by HET (36.6%). The proportion of active and resolved cases among individuals with non-German origin was higher than in people with German origin (8.8%, versus 4.3%; p < 0.001) and the proportion of resolved HCV infections lower (27.8% versus 34.0%; p = 0.027). Conclusions The proportion of resolved HCV infections among people newly diagnosed with HIV-1 increased after the introduction of DAAs in Germany. The high prevalence and the low proportion of resolved HCV infections reveal that unmet diagnostic and therapeutic needs exist among PWIDs. The higher proportion of active and resolved cases among individuals of non-German origin particularly requires greater public health attention.

Neutralizing antibody analysis against SARS-CoV-2 variants requires assays that rapidly adapt to protein mutations. Here, we present a protocol for monitoring neutralizing antibodies to various receptor-binding domains using the tANCHOR system. We describe steps for displaying variant receptor-binding domains on HeLa cells and producing tagged soluble angiotensin-converting enzyme 2 (ACE2). We then detail the procedures for establishing a cell-based ELISA to measure serum neutralization efficiency, using ACE2 competition as a readout. For complete details on the use and execution of this protocol, please refer to Ivanusic et al.1.

Monitoring specific antibodies derived from whole-cell immunization through cell-based ELISA methods poses challenges due to humoral responses against various cell proteins. In this report, we outline a technique involving pre-adsorption on cells to remove undesirable antibodies from immune serum. This step provides the subsequent monitoring of antibodies specific to the targeted antigen using a tANCHOR-based ELISA. Notably, this approach accelerates result acquisition, eliminating the necessity to purify the expressed antigen or obtain a customized peptide for coating assay plates.

Conventional neutralizing enzyme-linked immunosorbent assay (ELISA) systems for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mimic the protein-protein interaction between angiotensin-converting enzyme 2 (ACE2) and the receptor-binding domain (RBD). However, an easy and rapidly adaptative ELISA-based system for testing neutralizing antibodies against upcoming SARS-CoV-2 variants is urgently needed. In this study, we closed this gap by developing a tANCHOR-cell-based RBD neutralization assay that avoids time-consuming protein expression and purification followed by coating on ELISA plates. This cell-based assay can be rapidly adopted to monitor neutralizing antibodies (NAbs) against upcoming SARS-CoV-2 variants. We show that the results obtained with the tANCHOR-cell-based assay system strongly correlate with commercially available surrogate assays for testing NAbs. Moreover, this technique can directly measure binding between cell-surface-exposed RBDs and soluble ACE2. With this technique, the degree of antibody escape elicited by emerging SARS-CoV-2 variants in current vaccination regimens can be determined rapidly and reliably.

Enzyme-linked immunosorbent assay (ELISA) systems use plates coated with peptides or expressed and purified proteins to monitor immunoglobulins derived from patient serum. However, there is currently no easy, flexible, and fast adaptive ELISA-based system for testing antibodies directed against new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. In this study, we utilized the tANCHOR protein display system that provides a cell surface decorated with the receptor-binding domain (RBD) to monitor specific antibodies derived from SARS-CoV-2 convalescent and vaccinated individuals directed against it. To test sera from vaccinees or convalescent individuals, only the RBD coding sequence needs to be cloned in the tANCHOR vector system and transfected into HeLa cells. Time-consuming protein expression, isolation, and purification followed by coating assay plates are not necessary. With this technique, the immune evasion of new SARS-CoV-2 variants from current vaccination regimes can be examined quickly and reliably.

Lipopolysaccharide (LPS) contaminations may falsify immunological experiments and are crucial for pharmaceutical products because they cause life-threatening immune reactions. Here, we present interleukin-10 (IL-10) as a reliable marker to measure LPS contents when the readout of pro-inflammatory cytokines is not favored. This animal free source assay is able to detect LPS with a limit of detection (LOD) of 0.024 EU/ml by monitoring IL-10 secretions from isolated human peripheral blood mononuclear cells (PBMCs).

Successful induction of antibodies in model organisms like mice depends strongly on antigen design and delivery. New antigen designs for immunization are helpful for developing future therapeutic monoclonal antibodies (mAbs). One of the gold standards to induce antibodies in mice is to express and purify the antigen for vaccination. This is especially time-consuming when mAbs are needed rapidly. We closed this gap and used the display technology tetraspanin anchor to develop a reliable immunization technique without the need to purify the antigen. This technique is able to speed up the immunization step enormously and we have demonstrated that we were able to induce antibodies against different proteins with a focus on the receptor-binding domain of SARS-CoV-2 and the extracellular loop of canine cluster of differentiation 20 displayed on the surface of human cells.

Exosomes are small extracellular vesicles that are secreted from cells. To characterize exosome fraction marker proteins of the tetraspanin family in particular, CD9, CD63, and CD81 are routinely used. CD63 expression constructs were employed to investigate the influence of the large extracellular loop (LEL) of CD63 on sorting into exosomes. When the LEL of CD63 fused with mCherry was deleted, the protein was no longer found in the purified exosome fraction. This finding demonstrates the importance of the LEL sequence for the recruitment of CD63 into exosomes.

Time-lapse imaging provides an uninterrupted observation method that can lead to understanding protein dynamics. We previously developed a technique based on thin agar pads to keep the cells in focus during confocal laser scanning microscope imaging. Using this method, time-lapse imaging was employed to monitor CD63 fused to mCherry at the virological synapse (VS) during viral cluster transfer to acceptor cells of the human immunodeficiency virus 1 (HIV-1).

Human immunodeficiency virus type 1 (HIV-1) persists lifelong in infected individuals and has evolved unique strategies in order to evade the immune system. One of these strategies is the direct cell-to-cell spread of HIV-1. The formation of a virological synapse (VS) between donor and target cell is important for this process. Tetraspanins are cellular proteins that are actively involved in the formation of a VS. However, the molecular mechanisms of recruiting host proteins for the cell-cell transfer of particles to the VS remains unclear. Our study has mapped the binding site for the transmembrane envelope protein gp41 of HIV-1 within the large extracellular loop (LEL) of CD63 and showed that this interaction occurs predominantly at the VS between T cells where viral particles are transferred. Mutations within the highly conserved CCG motif of the tetraspanin superfamily abrogated recruiting of expressed HIV-1 GFP fused Gag core protein and CD63 to the VS. This demonstrates the biological significance of CD63 for enhanced formation of a VS. Since cell-cell spread of HIV-1 is a major route of persistent infection, these results highlight the central role of CD63 as a member of the tetraspanin superfamily during HIV-1 infection and pathogenesis.

A novel tool for the presentation of peptides and small proteins on the surface of human cells has been developed. Our tANCHOR system utilizes tetraspanin anchors containing heterologous amino acid sequences inserted instead of the large extracellular loop. This technology allows a highly effective extracellular display of epitopes for antibody binding studies and many other potential applications.

One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic.

Immunosuppression by retroviruses including the human immunodeficiency virus-1 (HIV-1) is well known, however the mechanisms how retroviruses induce this immunosuppression is not fully investigated. It was shown that non-infectious retroviral particles as well as retroviral or recombinant retroviral transmembrane envelope (TM) proteins demonstrated immunosuppressive properties. The same was shown for peptides corresponding to a highly conserved domain in the TM protein. This domain is called immunosuppressive (ISU) domain and it induces modulation of the cytokine release of peripheral blood mononuclear cells (PBMCs) from healthy donors. In addition, it changes the gene expression of these cells. Common indications for the immunosuppressive activity were tumour growth in vivo and interleukin-10 (IL-10) release from human PBMCs in vitro. Single mutations in the ISU domain abrogated the immunosuppressive activity. In order to develop a new model system for the expression of the ISU domain and presentation to PBMCs which is not prone to possible endotoxin contaminations, two expression systems were developed. In the first system, designated pOUT, retroviral proteins containing the ISU domain were expressed and released into the cell culture medium, and in the second system, tANCHOR, the ISU domain was presented by a tetraspanin-anchored sequence on the cell surface of human cells. Both systems were exploited to express the wild-type (wt) ISU domains of HIV-1, of the porcine endogenous retrovirus (PERV) and of the murine leukaemia virus (MuLV) as well as to express mutants (mut) of these ISU domains. PERV is of special interest in the context of virus safety of xenotransplantation using pig organs. Expression of the TM proteins was demonstrated by confocal laser scanning microscopy, ELISA and Western blot analyses using specific antibodies. However, when cells expressing and releasing the ISU were co-incubated with human PBMCs, no increased production of IL-10 was observed when compared with the mutants. Similar results were obtained when the released TM proteins were concentrated by immunoprecipitation and added to PBMCs. We suggest that the absence of IL-10 induction can be explained by a low amount of protein, by the lack of a biologically active conformation or the absence of additional factors.

Dynamic processes of cells can be best monitored when living cells are analyzed by imaging. While it is easy to observe adherent living cells it has been extremely challenging to analyze suspension cells. This cell type floats freely in the culture dish, and it is only a question of time when the focus or the observation field is lost. In order to keep the cells in focus, an easy and inexpensive method allowing the observation of living suspension cells during confocal laser scanning microscope imaging was developed.

Porcine cytomegalovirus (PCMV) infection is widely prevalent among pigs, and PCMV is one of the viruses which may be transmitted during xenotransplantation using pig cells, tissues, or organs. While human cytomegalovirus (HCMV) is a major risk factor for allotransplantation, it is still unclear whether PCMV is able to infect human cells or pose a risk for xenotransplantation. Previously, it was shown that transmission of PCMV after pig kidney to non-human primate transplantations resulted in a significantly reduced survival time of the transplanted organ. To detect PCMV, PCR-based and immunological methods were used. Screening of pigs by Western blot analyses using recombinant viral proteins revealed up to 100% of the tested animals to be infected. When the same method was applied to screen human sera for PCMV-reactive antibodies, positive Western blot results were obtained in butchers and workers in the meat industry as well as in normal blood donors. To exclude an infection of humans with PCMV, the sera were further investigated. PCMV is closely related to human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7), and a sequence alignment of glycoprotein B suggests that the antibodies may cross-react with identical epitope sequences. HCMV is not related with PCMV, and no correlation between antibody reactivity against PCMV and HCMV was detected. These data indicate that antibodies against PCMV found in humans are cross-reactive antibodies against HHV-6.

Porcine cytomegalovirus (PCMV) may be harmful for human recipients if xenotransplantation using pig cell, tissue or organ will be performed transmitting the virus from donor pigs to human recipients. PCMV is widespread in pigs and closely related to human pathogenic herpesviruses, however there are no data concerning infection of humans. In contrast, recently it had been shown that transplantation of organs from pigs infected with PCMV into non-human primate recipients resulted in a significant reduction of the survival time compared with the transplantation of organs from uninfected pigs. To prevent transmission of PCMV in future pig to human xenotransplantations, sensitive and specific detection methods should be used. Here a new Western blot assay using recombinant proteins corresponding to two domains of the glycoprotein gB of PCMV is described. With this assay, the presence of PCMV-specific antibodies in different pig breeds was analysed. Antibodies were detected in a high percentage of animals, in one breed up to 85%.

This unit provides a guide and detailed protocol for studying membrane protein-protein interactions (PPI) using the acceptor-sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET-PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET-PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor. The correlative application of FRET and PLA combines two powerful tools for monitoring PPI, yielding results that are more reliable than with either technique alone. © 2016 by John Wiley & Sons, Inc.