Single Chain Trimer Research Articles

Induction of T-cell response by a DNA vaccine encoding a novel HLA-A*0201 severe acute respiratory syndrome coronavirus epitope.

The severe acute respiratory syndrome coronavirus nucleocapsid protein (SARS-CoV N) is one of the major targets for SARS vaccine due to its high potency in triggering immune responses. In this study, we have identified a novel HLA-A*0201 restricted epitope, N220 (LALLLLDRL), of the SARS-CoV N-protein through bioinformatics analysis. The N-protein peptide N220 shows a high binding affinity towards human MHC class I in T2-cells, and is capable of activating cytotoxic T-cells in human peripheral blood mononuclear cells (PBMCs). The application of using the N220 peptide sequence with a single-chain-trimer (SCT) approach to produce a potential DNA vaccine candidate was investigated in HLA-A2.1Kb transgenic mice. Cytotoxicity assay clearly showed that the T-cells obtained from the vaccinated animals were able to kill the N-protein expressing cells with a cytotoxicity level of 86% in an effector cells/target cells ratio of 81:1 one week after the last vaccination, which is significantly higher than other N-protein peptides previously described. The novel immunogenic N-protein peptide revealed in the present study provides valuable information for therapeutic SARS vaccine design.

Disulfide Bond Engineering to Trap Peptides in the MHC Class I Binding Groove

Immunodominant peptides in CD8 T cell responses to pathogens and tumors are not always tight binders to MHC class I molecules. Furthermore, antigenic peptides that bind weakly to the MHC can be problematic when designing vaccines to elicit CD8 T cells in vivo or for the production of MHC multimers for enumerating pathogen-specific T cells in vitro. Thus, to enhance peptide binding to MHC class I, we have engineered a disulfide bond to trap antigenic peptides into the binding groove of murine MHC class I molecules expressed as single-chain trimers or SCTs. These SCTs with disulfide traps, termed dtSCTs, oxidized properly in the endoplasmic reticulum, transited to the cell surface, and were recognized by T cells. Introducing a disulfide trap created remarkably tenacious MHC/peptide complexes because the peptide moiety of the dtSCT was not displaced by high-affinity competitor peptides, even when relatively weak binding peptides were incorporated into the dtSCT. This technology promises to be useful for DNA vaccination to elicit CD8 T cells, in vivo study of CD8 T cell development, and construction of multivalent MHC/peptide reagents for the enumeration and tracking of T cells-particularly when the antigenic peptide has relatively weak affinity for the MHC.

Monoclonal antibodies to HLA class I restricted influenza epitopes (93.8)

Abstract The immune response to viruses involves the presentation of peptide epitopes in the context of the human leukocyte antigen (HLA) class I molecule. The HLA class I molecule consists of a heavy chain, a light chain, and a peptide of 9–10 amino acids that is positioned in a groove formed by the class I heavy chain. Identification of HLA class I-restricted peptide epitopes that distinguish a virus-infected cell from a non-infected cell is positioned to facilitate studies of antigen presentation, viral diagnostics, and the design of viral vaccines/therapeutics. Recently, we have directly discovered epitopes presented by the class I HLA of influenza-infected cells. Using HLA tetramers and single chain trimers (SCT) as immunogens, we have produced monoclonal antibodies (mAb) to HLA class I molecules carrying viral peptide epitopes. The peptide/HLA specificity of the mAbs was confirmed by ELISA and by flow cytometry. The results of these experiments demonstrate that mAb specific for HLA/influenza peptide epitopes could be useful for studying the dynamics of viral epitope presentation and may represent a tool for targeting viral infected cells.

DNA Vaccines Encoding Ii-PADRE Generates Potent PADRE-specific CD4+ T-Cell Immune Responses and Enhances Vaccine Potency

DNA Vaccines Encoding Ii-PADRE Generates Potent PADRE-specific CD4+ T-Cell Immune Responses and Enhances Vaccine Potency

Transgenic expression of HLA‐E single chain trimer protects porcine endothelial cells against human natural killer cell‐mediated cytotoxicity

The susceptibility of porcine endothelial cells (pEC) to human natural killer (NK) cells is related to the failure of human major histocompatibility complex (MHC)-specific killer inhibitory receptors to recognize porcine MHC class I molecules. The aims of this study were (i) to assess the protection of pEC against xenogeneic NK-mediated cytotoxicity afforded by the stable expression of HLA-E single chain trimers (SCT) composed of a canonical HLA-E binding peptide antigen, VMAPRTLIL, the mature human beta2-microglobulin, and the mature HLA-E heavy chain, and (ii) to test whether HLA-E expression on pEC and porcine lymphoblastoid cells affects the adhesion of human NK cells. Porcine EC lines expressing different levels of HLA-E SCT were generated by Ca(2)PO(4)-transfection followed by limiting dilution cloning. Surface expression of HLA-E was measured by flow cytometry. Susceptibility of transfected pEC lines against human NK cells was tested in (51)Cr-release cytotoxicity assays. Interactions between human NK cells and HLA-E positive pEC or porcine lymphoblastoid cells were further addressed in adhesion and conjugation assays. The level of protection of pEC from human NK-mediated cytotoxicity correlated with the intensity of surface HLA-E expression. Furthermore, the HLA-E SCT-mediated protection was specifically reversed by blocking the HLA-E specific NK inhibitory receptor CD94/NKG2A. HLA-E expression does neither affect the adhesion of human NK cells to pEC nor the heteroconjugate formation between human NK and porcine 13271.10 cells. Stable surface expression of HLA-E on pEC was achieved in the absence of extrinsic peptide pulsing and provided partial protection from human NK cytotoxicity. Though insufficient to inhibit xenogeneic NK cell reactivity completely, transgenic HLA-E expression on pig organs might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.

Hepatitis B virus core antigen epitopes presented by HLA‐A2 single‐chain trimers induce functional epitope‐specific CD8+ T‐cell responses in HLA‐A2·1/Kb transgenic mice

The potency of CD8+ cytotoxic T lymphocyte (CTL) responses toward core antigen has been shown to affect the outcomes of hepatitis B virus (HBV) infection. Since single-chain trimers (SCT) composed of peptide epitope beta2-microglobulin (beta2m) and major histocompatibility complex (MHC) class I heavy chain covalently linked together in a single molecule have been shown to stimulate efficient CTL responses, we investigated the properties of human leucocyte antigen (HLA)-A2 SCTs encoding the HBV core antigen (HBcAg) epitopes C(18-27) and C(107-115). Transfection of NIH-3T3 cells with pcDNA3.0-SCT-C(18-27) and SCT-C(107-115) leads to stable presentation of HBcAg epitopes at the cell surface. HLA-A2.1/Kb transgenic mice vaccinated with the SCT constructs, either as a DNA vaccine alone or followed by a boost with recombinant vaccinia virus, were shown to generate HBcAg-specific CTL responses by enzyme-linked immunospot assay (ELISPOT) and in vitro interferon-gamma release experiments. HBcAg-specific CTLs from vaccinated HLA-A2.1/Kb transgenic mice were able to inhibit HBV surface and e antigen expression as indicated by HepG2.2.15 cells. Our data indicate that a DNA vaccine encoding a human HLA-A2 SCT with HBV epitopes can lead to stable, enhanced HBV core antigen presentation, and may be useful for the control of HBV infection in HLA-A2-positive HBV carriers.

Preparation of MHC class I -antigenic peptide single-chain trimer and its application in immunotherapy for cancers

主要组织相容性复合物(MHC)Ⅰ类分子-抗原肽单链三聚体(SCT)由MHCⅠ类分子的重链、β2微球蛋白及抗原肽分子用接头相连接折叠形成.SCT能有效地维持其共价结构,并稳定地在细胞表面表达,能更有效地激活抗原特异性的CD8+细胞毒性T淋巴细胞,从而能更有效地清除病原体感染和恶性肿瘤细胞。

A DNA vaccine encoding a single-chain trimer of HLA-A2 linked to human mesothelin peptide generates anti-tumor effects against human mesothelin-expressing tumors

Mesothelin is highly expressed in a majority of ovarian cancer cells and is expressed at low levels in normal cells. Therefore, mesothelin represents a potential target antigen for ovarian cancer vaccine development. DNA vaccines employing single-chain trimers (SCT) have been shown to bypass antigen processing and presentation and result in significant enhancement of DNA vaccine potency. In the current study, we created a DNA vaccine employing an SCT targeting human mesothelin and characterized the ensuing antigen-specific CD8 + T cell-mediated immune responses and anti-tumor effects against human mesothelin-expressing tumors in HLA-A2 transgenic mice. Our results showed that vaccination with DNA employing an SCT of HLA-A2 linked to human mesothelin epitope aa540–549 (pcDNA3–Hmeso540–β2m–A2) generated strong human mesothelin peptide (aa540–549)-specific CD8 + T cell immune responses in HLA-A2 transgenic mice. Vaccination with pcDNA3–Hmeso540–β2m–A2 prevented the growth of HLA-A2 positive human mesothelin-expressing tumor cell lines in HLA-A2 transgenic mice in contrast to vaccination with DNA encoding SCT linked to OVA CTL epitope. Thus, the employment of SCT of HLA-A2 linked to the human mesothelin epitope aa540–549 represents a potential opportunity for the clinical translation of DNA vaccines against human mesothelin-expressing tumors, particularly ovarian cancer cells.

Do Close Encounters Exclude Large Proteins?

Signaling from the T cell antigen receptor (TCR) is initiated (TCR triggering) when the TCR is bound by peptide presented by the major histocompatibility antigens (pMHC) on antigen-presenting cells (APCs). Whereas the TCR-pMHC interaction spans an intermembrane distance of about 14 nm (as do various receptor-ligand pairs that contribute to TCR triggering), receptor tyrosine phosphatases such as CD45 and CD148, which inhibit TCR triggering, have much larger ectodomains. Choudhuri et al . created murine pMHC class I complexes as single-chain trimer (SCT) fusion proteins containing the ovalbumin-derived peptide SIINFEKL (pOVA), β2-microglobulin, and the H-2K b MHC class I heavy chain. SCTs were elongated by including two, three, or four immunoglobulin superfamily domains to test the hypothesis that TCR triggering involves size-dependent exclusion of proteins with large ectodomains and thus depends on the intermembrane distance spanned by the TCR-pMHC. When expressed in Chinese hamster ovary (CHO) cells, elongated SCTs were less effective at activating a T cell hybridoma (B3Z cells) or CD8 + T cells that specifically recognized pOVA-H2K b . Transmission electron microscopy (TEM) showed that the mean intermembrane distance between B3Z cells and APCs expressing the elongated SCT containing four immunoglobulin superfamily domains (SCT-CD4) was substantially greater (17.8 nm) than that with cells expressing unelongated SCT (13.1 nm). Moreover, an early depletion of CD45 from the T cell-APC interface seen when T cells were incubated with APCs expressing SCT was not apparent when T cells were incubated with APCs expressing SCT-CD4. Similar results were obtained with an artificial TCR-ligand system. Thus, the authors conclude that TCR triggering depends on close contact between the T cell and the APC and the size-dependent segregation of proteins with large ectodomains from the T cell-APC interface. K. Choudhuri, D. Wiseman, M. H. Brown, K. Gould, P. A. van der Merwe, T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand. Nature 436 , 578-582 (2005). [PubMed]

Cancer immunotherapy using a DNA vaccine encoding a single-chain trimer of MHC class I linked to an HPV-16 E6 immunodominant CTL epitope

The potency of DNA vaccines may be affected by the efficiency of intracellular processing and MHC class I presentation of encoded antigens. Since a single-chain trimer (SCT) composed of peptide, beta2-microglobulin (beta2m), and MHC class I heavy chain has been shown to bypass antigen processing and lead to stable presentation of peptides, we investigated the efficacy of a DNA vaccine encoding a SCT composed of an immunodominant CTL epitope of human papillomavirus type 16 (HPV-16) E6 antigen, beta2m, and H-2Kb MHC class I heavy chain (pIRES-E6-beta2m-Kb). Transfection of 293 cells with pIRES-E6-beta2m-Kb can bypass antigen processing and lead to stable presentation of E6 peptide. Furthermore, C57BL/6 mice vaccinated with pIRES-E6-beta2m-Kb exhibited significantly increased E6 peptide-specific CD8+ T-cell immune responses compared to mice vaccinated with DNA encoding wild-type E6. Most importantly, 100% of mice vaccinated with pIRES-E6-beta2m-Kb DNA were protected against a lethal challenge of E6-expressing TC-1 tumor cells. In contrast, all mice vaccinated with wild-type E6 DNA or control plasmid DNA grew tumors. Our data indicate that a DNA vaccine encoding a SCT can lead to stable enhanced MHC class I presentation of encoded antigenic peptide and may be useful for improving DNA vaccine potency to control tumors or infectious diseases.

An HLA-E single chain trimer inhibits human NK cell reactivity towards porcine cells

HLA-E, when expressed by pig cells, could alleviate human natural killer (NK) cell-mediated rejection of porcine xenografts by providing a potent inhibitory ligand for human NK cells expressing CD94/NKG2A. Yet cell-surface expression of HLA-E on porcine epithelial (LLC-PK1) cells was not observed after transfection with an expression vector harboring HLA-E alone or in combination with an expression vector containing human β2m. A single chain trimer (SCT) of HLA-E consisting of, in the following order (from N- to C-terminus), the leader peptide of human β2m, VMAPRTLIL (an HLA-E-binding peptide), a 15 amino acid linker, mature human β2m, a 20 amino acid linker, and mature HLA-E heavy chain was engineered. Cell-surface expression and correct folding of HLA-E SCT was shown by FACS analyses of stably transfected LLC-PK1 cells. Untransfected LLC-PK1 cells were readily lysed by the NK cell lines NKL and NK-92, while LLC-PK1 cells expressing HLA-E SCT were almost completely protected. In addition, the HLA-E SCT recapitulates the peptide dependent properties of normal HLA-E trimeric complexes in that an HLA-E SCT with an hsp60 derived peptide, though expressed at the cell-surface, did not inhibit NK cell-mediated lysis. The HLA-E SCT, which conferred protection against NK cell-mediated killing, also inhibited NK cell IFN-γ secretion elicited by co-culture of NKL cells with LLC-PK1 cells. Thus, HLA-E SCT, in which all three components of a normal HLA-E protein complex are in one polypeptide chain, is immunologically functional as it is able to modulate NK cell cytotoxicity and cytokine secretion.

Applications of Major Histocompatibility Complex Class I Molecules Expressed as Single Chains

Generation of CD8 T-cell responses to pathogens and tumors requires optimal expression of class I major histocompatibility complex/peptide complexes, which, in turn, is dependent on host cellular processing events and subject to interference by pathogens. To create a stable structure that is more immunogenic and resistant to immune evasion pathways, we have engineered class I molecules as single-chain trimers (SCTs), with flexible linkers connecting peptide, beta2m, and heavy chain. Herein we extend our earlier studies with SCTs to the K(b) ligand derived from vesicular stomatitis virus (VSV) to characterize further SCTs as probes of immune function as well as their potential in immunotherapy. The VSVp-beta2m-K(b) SCTs were remarkably stable at the cell surface, and immunization with DNA encoding SCTs elicited complex-specific antibody. In addition, SCTs were detected by cytotoxic T-lymphocytes specific for the native molecule, and the covalently bound peptide was highly resistant to displacement by exogenous peptide. SCTs can also prime CD8 T-cells in vivo that recognize the native molecule. Furthermore, SCTs were resistant to downregulation by the immune evasion protein mK3 of gamma herpesvirus 68. Moreover, owing to their preassembled nature, SCTs should be resistant to other immune evasion proteins that restrict peptide supply. Thus, SCTs possess therapeutic potential both for prophylactic treatment and for the treatment of ongoing infection.

Immunosuppressive properties of anti-CD3 single-chain Fv and diabody.

The mouse anti-human CD3 monoclonal antibody OKT3 is a potent immunosuppressive agent used in clinical transplantation. However, OKT3 therapy is associated with unpleasant and often serious side effects which appear to result from cytokine release, complement activation and a human anti-mouse antibody (HAMA) response. To decrease these adverse side effects, we constructed antibody fragments comprising OKT3 variable domains without any constant domains. Single-chain Fv (scFv) monomers, dimers and trimers were generated by changing the linker length between the V(H) and V(L) domains. The linkers used were the natural extensions of the V(H) into the C(H)1 domain. The dimeric molecules (diabodies) demonstrated the best CD3-binding activity. The diabody with the six amino acid linker was produced in bacteria with a tenfold higher yield than other scFvs and possessed CD3-binding affinity approaching that of the parental mAb. In contrast to OKT3 mAb, the anti-CD3 diabody and scFv monomer did not cause any T-cell activation and cytokine release in vitro, while demonstrating CD3 modulation. In mixed lymphocyte cultures, both diabody and scFv, but not the monoclonal antibody OKT3, were able to suppress T-cell activation and secretion of IL-2 and IFN-gamma in a dose-dependent manner. The anti-CD3 diabody may provide a potent immunosuppressive drug with low toxicity and immunogenicity.

Enhanced Immune Presentation of a Single-chain Major Histocompatibility Complex Class I Molecule Engineered to Optimize Linkage of a C-terminally Extended Peptide

Major histocompatibility complex class I molecules can be expressed as single polypeptides wherein the antigenic peptide, beta2-microglobulin, and heavy chain are attached by flexible linkers. These molecules, single-chain trimers (SCTs), are remarkably stable at the cell surface compared with native (noncovalently attached) class I molecules. In this study, we used a structure-based approach to engineer an F pocket variant SCT of the murine class I molecule Kb that presents the SIINFEKL epitope of ovalbumin. Mutation of heavy chain residue Tyr84 (Y84A) in the SCT resulted in enhanced serological and cytolytic CD8 T cell recognition of the covalently linked peptide due to better accommodation of the linker extending from the C terminus of the peptide. These SCTs exhibit significant cell-surface stability, which we hypothesize is rendered by their ability to continuously and efficiently rebind the covalently attached peptide. In addition, we demonstrate that SCT technology can be applied to tetramer construction using recombinant SCTs expressed in Escherichia coli. SCT-based tetramers could have applications for the enumeration of T and natural killer cells that recognize peptide.class I complexes prone to dissociation.

Cutting Edge: Single-Chain Trimers of MHC Class I Molecules Form Stable Structures That Potently Stimulate Antigen-Specific T Cells and B Cells

We report in this work the expression and characterization of class I molecules expressed as single-chain trimers consisting of an antigenic peptide-spacer-beta(2)-microglobulin-spacer H chain. Our results indicate that these single-chain constructs assemble efficiently, maintain their covalent structure, and are unusually stable at the cell surface. Consequently, these constructs are at least 1000-fold less accessible to exogenous peptide than class I molecules loaded with endogenous peptides, and they are potent simulators of peptide-specific CTL and Abs. Our combined findings suggest that single-chain trimers may have applications as DNA vaccines against virus infection or tumors.