Enhanced stability of a chimeric hepatitis B core antigen virus-like-particle (HBcAg-VLP) by a C-terminal linker-hexahistidine-peptide.

BACKGROUND: Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. RESULTS: The effects of a carboxy-terminal polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. CONCLUSIONS: These findings establish the genetically simply transferable C-terminal polyhistidine-peptide as a general stabilizing element for chimeric HBcAg-VLPs to increase their suitability.

Expression and translation of the COX-1b gene in human cells--no evidence of generation of COX-1b protein.

Cyclooxygenase 1b (COX-1b) is a splice variant of COX-1, containing a retained intron 1 within the signal peptide sequence. COX-1b mRNA is found in many species, but the existence of a functionally active protein, which is possibly related to different species-dependent lengths of intron 1, is controversially discussed. The human intron 1 comprises 94 bp, and the resulting frameshift at the intron 1-exon 2 junction creates a premature stop codon. Nevertheless, full-length human COX-1b protein expression, including translated intron 1 and the signal peptide, has been reported and was explained by a frameshift repair. In this study, the fate of COX-1b mRNA in a human overexpression system is analyzed. Independent of the hypothetical frameshift repair mechanism, the splicing of the COX-1b intron 1, resulting in COX-1 mRNA and removal of the signal peptide during protein maturation, with subsequent generation of a COX-1 protein is demonstrated.

Ensembles of Hydrophobicity Scales as Potent Classifiers for Chimeric Virus-Like Particle Solubility - An Amino Acid Sequence-Based Machine Learning Approach.

Virus-like particles (VLPs) are protein-based nanoscale structures that show high potential as immunotherapeutics or cargo delivery vehicles. Chimeric VLPs are decorated with foreign peptides resulting in structures that confer immune responses against the displayed epitope. However, insertion of foreign sequences often results in insoluble proteins, calling for methods capable of assessing a VLP candidate's solubility in silico. The prediction of VLP solubility requires a model that can identify critical hydrophobicity-related parameters, distinguishing between VLP-forming aggregation and aggregation leading to insoluble virus protein clusters. Therefore, we developed and implemented a soft ensemble vote classifier (sEVC) framework based on chimeric hepatitis B core antigen (HBcAg) amino acid sequences and 91 publicly available hydrophobicity scales. Based on each hydrophobicity scale, an individual decision tree was induced as classifier in the sEVC. An embedded feature selection algorithm and stratified sampling proved beneficial for model construction. With a learning experiment, model performance in the space of model training set size and number of included classifiers in the sEVC was explored. Additionally, seven models were created from training data of 24-384 chimeric HBcAg constructs, which were validated by 100-fold Monte Carlo cross-validation. The models predicted external test sets of 184-544 chimeric HBcAg constructs. Best models showed a Matthew's correlation coefficient of >0.6 on the validation and the external test set. Feature selection was evaluated for classifiers with best and worst performance in the chimeric HBcAg VLP solubility scenario. Analysis of the associated hydrophobicity scales allowed for retrieval of biological information related to the mechanistic backgrounds of VLP solubility, suggesting a special role of arginine for VLP assembly and solubility. In the future, the developed sEVC could further be applied to hydrophobicity-related problems in other domains, such as monoclonal antibodies.

Optimization of a Soft Ensemble Vote Classifier for the Prediction of Chimeric Virus-Like Particle Solubility and Other Biophysical Properties.

Chimeric virus-like particles (cVLPs) are protein-based nanostructures applied as investigational vaccines against infectious diseases, cancer, and immunological disorders. Low solubility of cVLP vaccine candidates is a challenge that can prevent development of these very substances. Solubility of cVLPs is typically assessed empirically, leading to high time and material requirements. Prediction of cVLP solubility in silico can aid in reducing this effort. Protein aggregation by hydrophobic interaction is an important factor driving protein insolubility. In this article, a recently developed soft ensemble vote classifier (sEVC) for the prediction of cVLP solubility was used based on 91 literature amino acid hydrophobicity scales. Optimization algorithms were developed to boost model performance, and the model was redesigned as a regression tool for ammonium sulfate concentration required for cVLP precipitation. The present dataset consists of 568 cVLPs, created by insertion of 71 different peptide sequences using eight different insertion strategies. Two optimization algorithms were developed that (I) modified the sEVC with regard to systematic misclassification based on the different insertion strategies, and (II) modified the amino acid hydrophobicity scale tables to improve classification. The second algorithm was additionally used to synthesize scales from random vectors. Compared to the unmodified model, Matthew's Correlation Coefficient (MCC), and accuracy of the test set predictions could be elevated from 0.63 and 0.81 to 0.77 and 0.88, respectively, for the best models. This improved performance compared to literature scales was suggested to be due to a decreased correlation between synthesized scales. In these, tryptophan was identified as the most hydrophobic amino acid, i.e., the amino acid most problematic for cVLP solubility, supported by previous literature findings. As a case study, the sEVC was redesigned as a regression tool and applied to determine ammonium sulfate concentrations for the precipitation of cVLPs. This was evaluated with a small dataset of ten cVLPs resulting in an R 2 of 0.69. In summary, we propose optimization algorithms that improve sEVC model performance for the prediction of cVLP solubility, allow for the synthesis of amino acid scale tables, and further evaluate the sEVC as regression tool to predict cVLP-precipitating ammonium sulfate concentrations.

Displaying Tetra-Membrane Spanning Claudins on Enveloped Virus-Like Particles for Cancer Immunotherapy.

Virus-like particles (VLPs) displaying foreign antigens have become an important tool in vaccination including the induction of immune responses against self-antigens. Claudin 6 (CLDN6) has been identified as tumor-associated antigen and is therefore a potential target for tumor vaccination strategies. However, as tetra-membrane spanning protein its incorporation into VLPs while preserving a native fold is challenging. Here, we attempted the incorporation of a panel of engineered CLDN6 variants into the membrane of retrovirus-derived VLPs. Interestingly, wild-type CLDN6 revealed the most efficient display. VLPs presenting CLDN6 or CLDN9 derived from different donor species were produced and preservation of their native confirmation was demonstrated by antibody binding assays. VLPs displaying murine CLDN6 were used to immunize mice. Antibodies recognizing native CLDN6 as displayed on cell surfaces and mediating complement-dependent cytotoxicity were elicited in vaccinated animals. The data suggest applications of CLDN6 displaying VLPs in cancer immunotherapy.

Antigen-specific oncolytic MV-based tumor vaccines through presentation of selected tumor-associated antigens on infected cells or virus-like particles.

Recombinant vaccine strain-derived measles virus (MV) is clinically tested both as vaccine platform to protect against other pathogens and as oncolytic virus for tumor treatment. To investigate the potential synergism in anti-tumoral efficacy of oncolytic and vaccine properties, we chose Ovalbumin and an ideal tumor antigen, claudin-6, for pre-clinical proof of concept. To enhance immunogenicity, both antigens were presented by retroviral virus-like particle produced in situ during MV-infection. All recombinant MV revealed normal growths, genetic stability, and proper expression and presentation of both antigens. Potent antigen-specific humoral and cellular immunity were found in immunized MV-susceptible IFNAR-/--CD46Ge mice. These immune responses significantly inhibited metastasis formation or increased therapeutic efficacy compared to control MV in respective novel in vivo tumor models using syngeneic B16-hCD46/mCLDN6 murine melanoma cells. These data indicate the potential of MV to trigger selected tumor antigen-specific immune responses on top of direct tumor lysis for enhanced efficacy.

Expression profiling of autoimmune regulator AIRE mRNA in a comprehensive set of human normal and neoplastic tissues.

Defects in the autoimmune regulator (AIRE) gene cause the monogenic autoimmune disease autoimmune polyendocrinopathy syndrome type 1 (APS-1), which is characterized by a loss of self-tolerance to multiple organs. In concordance with its role in immune tolerance, AIRE is strongly expressed in medullary thymic epithelial cells (mTECs). Data on mechanisms controlling AIRE activation and the expression of this gene in other tissues are fragmentary and controversial. We report here AIRE mRNA expression profiling of a large set of normal human tissues and cells, tumor specimen and methylation deficient cell lines. On this broad data basis we found that AIRE mRNA expression is confined to mTECs in thymus and to lymph node tissue and that DNA hypomethylation contributes to transcriptional control of this gene.

Regulation of the expression of mouse TAP-associated glycoprotein (tapasin) by cytokines.

The expression of antigen presenting MHC class I molecules can be enhanced through cytokines, e.g. upon infection with bacteria or viruses, either directly by enhancing class I gene transcription or by increasing the amounts of accessory proteins of the loading complex. Tapasin plays a significant role in the peptide loading of class I molecules. Here, we describe recognition motifs of cytokine inducible transcription factors in the promoter region of the mouse tapasin gene, most of them clustered within the 140 base pairs upstream of the start codon. Tapasin mRNA was strongly induced in vivo after infection with the facultatively intracellular bacterium Listeria monocytogenes in an IFN-gamma-dependent fashion. Accordingly, both tapasin mRNA and protein were strongly induced in a time and dose dependent manner in embryonic fibroblasts treated with the cytokines IFN-gamma and IFN-beta, and weakly induced after treatment with TNF-alpha. Co-stimulation of tapasin by TNF-alpha and IFN-gamma resulted in a weak synergistic effect. Using fibroblasts either lacking IRF-1 or inhibited in protein synthesis we show that secondary transcription factors are necessary for a maximal stimulation of tapasin expression upon IFN-gamma stimulation. The sequential induction of TAP1, LMP2, and tapasin before the stimulated expression of class I heavy chain is discussed.

Highly specific auto-antibodies against claudin-18 isoform 2 induced by a chimeric HBcAg virus-like particle vaccine kill tumor cells and inhibit the growth of lung metastases.

Strategies for antibody-mediated cancer immunotherapy, such as active immunization with virus-like particle (VLP)-based vaccines, are gaining increasing attention. We developed chimeric hepatitis B virus core antigen (HBcAg)-VLPs that display a surface epitope of the highly selective tumor-associated cell lineage marker claudin-18 isoform 2 (CLDN18.2) flanked by a mobility-increasing linker. Auto-antibodies elicited by immunization with these chimeric HBcAg-VLPs in 2 relevant species (mouse and rabbit) bind with high precision to native CLDN18.2 at physiologic densities on the surface of living cells but not to the corresponding epitope of the CLDN18.1 splice variant that differs by merely one amino acid. The induced auto-antibodies are capable of efficiently killing CLDN18.2 expressing cells in vitro by complement-dependent and antibody-dependent cell-mediated cytotoxicity. Moreover, they provide partial protective immunity against the challenge of mice with syngeneic tumor cells stably expressing CLDN18.2. Our study provides a first proof-of-concept that immunization combining VLPs as antigen carriers with specific conformational epitopes of a highly selective differentiation antigen may elicit auto-antibodies with high cytocidal and tumoricidal potential.

A giant GTPase, very large inducible GTPase-1, is inducible by IFNs.

The complex, partially overlapping, cellular responses to IFN type I (IFN-alpha and -beta) and IFN type II (IFN-gamma) involve several hundred genes that can be largely classified in terms of specific cellular programs functional in innate and adaptive immunity. Among these programs are previously unconsidered mechanisms of cell-autonomous resistance against various pathogens mediated by dedicated, largely novel families of GTPases. We report here the identification and characterization of a new GTPase family that contributes to the cellular response to both type I and type II IFNs. We name this family the very large inducible GTPases (VLIGs). The prototype VLIG, VLIG-1, is a strongly IFN-inducible, soluble, cytosolic and nuclear protein of 280 kDa. The open reading frame of VLIG-1 is encoded on a single very large exon, and outside the canonical GTP-binding motifs, sequence and structural prediction suggest a unique family without significant relationship to other known protein families. Within the GTPase superfamily the VLIG family is more closely related to IFN-inducible GTPases mediating cell-autonomous resistance than to other GTPase families. In addition, we provide evidence that VLIG-1 is polymorphic in mice of different genetic backgrounds and is a member of a small gene family on mouse chromosome 7 with a conserved homologue located on human chromosome 11.