Inactivated Pseudomonas PE(ΔIII) exotoxin fused to neutralizing epitopes of PEDV S proteins produces a specific immune response in mice
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ABSTRACT:
SUBMITTER: Sun L
PROVIDER: S-EPMC8497069 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Seven-mer phage random peptide libraries were panned against 2C10, a monoclonal antibody that showed neutralizing activities against PEDV. Recombinant M13 phages displaying the peptides SHRLP(Y/Q)(P/V) or GPRPVTH on the g3p minor coat protein showed strong binding affinity with 2C10 (70% and 30% of recovered phages, respectively) after multiple panning. Sequence analysis suggested that these peptides are similar with (1368)GPRLQPY(1374) found at the carboxy-terminal of the S protein. In neutralization inhibition assays, the two peptide motifs and a 24-mer synthetic peptide corresponding to the C-terminal endodomain of PEDV S protein were observed to compete for the antigen binding site of 2C10, as demonstrated by the loss or reduction of neutralizing activity of the monoclonal antibody. This new finding suggests that the newly discovered peptide motifs mimic a neutralizing epitope PEDV.
| S-EPMC7111992 | biostudies-literature
Project description:Effective adaptive immune responses depend on activation of CD4+ T cells via the presentation of antigen peptides in the context of major histocompatibility complex (MHC) class II. The structure of an antigen strongly influences its processing within the endolysosome and potentially controls the identity of peptides that are presented to T cells. A recombinant immunotoxin, comprising exotoxin A domain III (PE-III) from Pseudomonas aeruginosa and a cancer-specific antibody fragment, has been developed to manage cancer, but its effectiveness is limited by the induction of neutralizing antibodies. Here, we observed that this immunogenicity is substantially reduced by substituting six residues within PE-III. Although these substitutions targeted T-cell epitopes, we demonstrate that reduced conformational stability and protease resistance were responsible for the reduced antibody titer. Analysis of mouse T-cell responses coupled with biophysical studies on single-substitution versions of PE-III suggested that modest but comprehensible changes in T-cell priming can dramatically perturb antibody production. The most strongly responsive PE-III epitope was well-predicted by a structure-based algorithm. In summary, single-residue substitutions can drastically alter the processing and immunogenicity of PE-III but have only modest effects on CD4+ T-cell priming in mice. Our findings highlight the importance of structure-based processing constraints for accurate epitope prediction.
| S-EPMC6433056 | biostudies-literature
Project description:Impaired apoptosis is often a key element in tumor development. Therefore, drugs mimicking prosurvival antagonists offer promise as cancer therapeutics. When ABT-737, a BH3-only mimetic, was added to KB3-1 human cervical adenocarcinoma cells, we noted an induction of an endoplasmic reticulum (ER) stress response and the dislocation of ER luminal proteins, including chaperones, to the cell cytosol. Furthermore, when immunotoxin (antibody-toxin chimeric molecule) and ABT-737 combinations were added to cells, there was enhanced toxin-mediated inhibition of protein synthesis, consistent with enhanced translocation of toxin to the cytosol. A similar enhancement was not seen with thapsigargin, suggesting that ER stress alone was not responsible for enhanced translocation. Cytosol preparations from ABT-737-treated but not from thapsigargin-treated cells revealed the presence of greater amounts of processed 37-kDa toxin fragment compared with the addition of immunotoxin alone. As early as 4 hours after the addition of ABT-737 and immunotoxin, there was release of mitochondrial cytochrome c and activation of caspase-3/7 indicating that the combination caused apoptotic cell death. These results were reflected in decreased cellular ATP levels that were noted with combinations of ABT-737 and immunotoxin but not with either agent alone or with combinations of thapsigargin and immunotoxin. We conclude that ABT-737 increases ER permeability, promoting the dislocation of toxin from the ER to the cytosol resulting in early apoptotic cell death. These mechanistic insights suggest why this class of BH3-only mimetic synergizes in a particular way with Pseudomonas exotoxin-based immunotoxins.
| S-EPMC6258188 | biostudies-literature
Project description:Pseudomonas exotoxin A (PE) is cytotoxic for eukaryotic cells because it enters cells by receptor-mediated endocytosis, translocates to the cell cytosol and ADP-ribosylates elongation factor 2 (EF2). However, the interaction of this toxin with eukaryotic cells and the mechanism of PE-mediated cell death have not been extensively characterized. The feasibility of carrying out a genome-wide RNAi screen, makes Drosophila melanogaster S2 cells as a good model system to identify essential genes in PE-mediated cytotoxicity, provided a suitable multi-well assay is developed. Here, using the alamarBlue viability assay, we show that Drosophila S2 cells are sensitive to PE at picomolar concentrations and that toxin treatments provoke an increase in caspase activity. This prompted us to use RNAi to characterize the mechanism of cell death. Results indicated that PE-mediated death of S2 cells was dependent on the presence of diphthamide, the post translational modification of EF2, and on the presence of Drice, the terminal caspase of insect cells. RNAi to drice or chemical inhibition of caspase action by z-VAD-fmk protected cells from PE-mediated death. Protection from death by RNAi or z-VAD-fmk did not interfere with toxin delivery to the cytosol leading to inhibition of protein synthesis. Using a convenient alamarBlue assay, our data confirms the cytotoxicity of PE for S2 cells and establishes apoptosis as the mode of PE-mediated death. This confirms the suitability of Drosophila cells as a convenient and simple model to elucidate the role of specific genes and proteins required for PE action.
| S-EPMC3431163 | biostudies-literature
Project description:Since late 2010, outbreaks of porcine epidemic diarrhea (PED) have been reported in the swine industry in China. A variant PEDV strain that differs from strain CV777 causes prevalent PEDV infections which commercial vaccines based on CV777 cannot provide complete protection. In this study, we designed a new vaccine based on the epidemic PEDV strain AH2012/12, adjuvanted with flagellin, a mucosal adjuvant that induces mucosal and systemic production of IgA. Three groups of pregnant sows were immunized twice, with a 14-day interval, with PEDV adjuvanted with flagellin, PEDV alone, or PBS before farrowing, and newborn piglets from each group were selected and challenged with PEDV. Immunization with this vaccine elicited high levels of IgG, IgA, and neutralizing antibodies in the serum and colostrum of sows, and newborn piglets were protected against PEDV while suckling. This study should guide the prevention and control strategies for PEDV infection, thereby reducing the losses associated with this virus.
| S-EPMC7223252 | biostudies-literature
Project description:Rhinoviruses (RVs) are major causes of the common cold, but they can also trigger exacerbations of asthma. More than 160 different RV strains exist and can be classified into three genetic species (RV-A, RV-B and RV-C) which bind to different receptors on human cells including intracellular adhesion molecule 1 (ICAM-1), the low-density lipoprotein receptor (LDLR) or the cadherin-related family member 3 (CDHR3). Epitopes located in the RV capsid have mainly been determined for RV2, a minor-group RV-A strain binding to LDLR, and for RV14, a major-group RV-B strain binding to ICAM-1. In order to study epitopes involved in the neutralization of RV89, an ICAM-1-binding RV-A strain which is highly different from RV2 and RV14 in terms of receptor specificity and sequence, respectively, we analyzed the specificity and epitopes of a highly neutralizing antiserum using recombinantly produced RV89 capsid proteins (VP1, VP2, VP3 and VP4), recombinant fragments and synthetic overlapping peptides thereof. We found that the antiserum which neutralized in vitro RV89 infection up to a dilution of 1:24,000 reacted with the capsid proteins VP1 and VP2 but not with VP3 and VP4. The neutralizing antibodies recognized recombinant fragments comprising approximately 100 amino acids of the N- and C-terminus of VP1 and the middle part of VP2, in particular, three peptides which, according to molecular modeling based on the three-dimensional structure of RV16, were surface-exposed on the viral capsid. Two recombinant fusion proteins containing the identified peptides fused to hepatitis B (HBV)-derived preS as a carrier protein induced upon immunization of rabbits antibodies capable of neutralizing in vitro RV89 infections. Interestingly, the virus-neutralizing epitopes determined for RV89 corresponded to those determined for minor-group RV2 binding to LDL and major-group RV14 belonging to the RV-B species, which are highly different from RV89. Our results indicate that highly different RV strains, even when reacting with different receptors, seem to engage similar parts of their capsid in the infection process. These results may be important for the design of active and passive immunization strategies for RV.
| S-EPMC9100655 | biostudies-literature
Project description:The ongoing coronavirus disease 2019 pandemic has raised concerns about the risk of re-infection. Non-neutralizing epitopes are one of the major reasons for antibody-dependent enhancement. Past studies on the ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have revealed an infectivity-enhancing site on the ancestral SARS-CoV-2 spike protein. However, infection enhancement associated with the SARS-CoV-2 Omicron strain remains elusive. In this study, we examined the antibodies induced by a multiple epitope-based vaccine, which showed infection enhancement for the Omicron strain but not for the ancestral SARS-CoV-2 or Delta strain. By examining the antibodies induced by single epitope-based vaccines, we identified a conserved epitope, IDf (450-469), with neutralizing activity against ancestral SARS-CoV-2, Delta, and Omicron. Although neutralizing epitopes are present in the multiple epitope-based vaccine, other immunodominant non-neutralizing epitopes such as IDg (480-499) can shade their neutralizing activity, leading to infection enhancement of Omicron. Our study provides up-to-date epitope information on SARS-CoV-2 variants to help design better vaccines or antibody-based therapeutics against future variants.
| S-EPMC9662650 | biostudies-literature
Project description:The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2. PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and protein translation assays demonstrate that the resulting fusions-supplied exogenously as genetic elements or purified protein fragments-had no significant catalytic activity or effect on protein synthesis individually but, in combination, catalyzed the ADP-ribosylation of eukaryotic elongation factor 2 and inhibited protein synthesis. Although complementing PE3 fragments are catalytically less efficient than intact PE3 in cell-free systems, co-expression in live cells transfected with transgenes encoding the toxin fusions inhibits protein synthesis and causes cell death comparably as intact PE3. Complementation of split PE3 offers a direct extension of the immunotoxin approach to generate bispecific agents that may be useful to target complex phenotypes.
| S-EPMC3997303 | biostudies-literature
Project description:Vaccinations can serve as an important preventive measure against the porcine epidemic diarrhea (PED) virus that currently threatens the swine industry. This study focuses on the development of a fusion protein vaccine, FliC99-mCOE, which combines the N-terminus of flagellin (FliC99) with a modified core neutralizing epitope (mCOE) of PEDV. In silico immunoinformatic analysis confirmed the construct's non-toxic, non-allergenic, and highly antigenic nature. Molecular docking and molecular dynamics (MD) simulations demonstrated FliC99-mCOE's strong binding to the TLR-5 immunological receptor. Repeated exposure simulations and immunological simulations suggested enhanced cell-mediated immunity. Both FliC99-mCOE and an inactivated PEDV vaccine were produced and tested in mice. The results from cell proliferation, ELISA, and neutralization assays indicated that FliC99-mCOE effectively stimulated cellular immunity and neutralized PEDV. We conclude that the FliC99-mCOE fusion protein may serve as a promising vaccine candidate against PEDV.
| S-EPMC10892538 | biostudies-literature
Project description:SARS-CoV-2 infection or vaccination produces neutralizing antibody responses that contribute to better clinical outcomes. The receptor binding domain (RBD) and the N-terminal domain (NTD) of the spike trimer (S) constitute the two major neutralizing targets for the antibody system. Neutralizing antibodies targeting the RBD bind to several different sites on this domain. In contrast, most neutralizing antibodies to NTD characterized to date bind to a single supersite, however these antibodies were obtained by methods that were not NTD specific. Here we use NTD specific probes to focus on anti-NTD memory B cells in a cohort of pre-omicron infected individuals some of which were also vaccinated. Of 275 NTD binding antibodies tested 103 neutralized at least one of three tested strains: Wuhan-Hu-1, Gamma, or PMS20, a synthetic variant which is extensively mutated in the NTD supersite. Among the 43 neutralizing antibodies that were further characterized, we found 6 complementation groups based on competition binding experiments. 58% targeted epitopes outside the NTD supersite, and 58% neutralized either Gamma or Omicron, but only 14% were broad neutralizers. Three of the broad neutralizers were characterized structurally. C1520 and C1791 recognize epitopes on opposite faces of the NTD with a distinct binding pose relative to previously described antibodies allowing for greater potency and cross-reactivity with 7 different variants including Beta, Delta, Gamma and Omicron. Antibody C1717 represents a previously uncharacterized class of NTD-directed antibodies that recognizes the viral membrane proximal side of the NTD and SD2 domain, leading to cross-neutralization of Beta, Gamma and Omicron. We conclude SARS-CoV-2 infection and/or Wuhan-Hu-1 mRNA vaccination produces a diverse collection of memory B cells that produce anti-NTD antibodies some of which can neutralize variants of concern. Rapid recruitment of these cells into the antibody secreting plasma cell compartment upon re-infection likely contributes to the relatively benign course of subsequent infections with SARS-CoV-2 variants including omicron.
| S-EPMC8820657 | biostudies-literature