Preparation of a capsaicinoids broad spectrum antibody and its application in non-enzyme immunoassay based on DMSNs@PDA@Pt.

Capsaicinoids (CPCs) is a special ingredient with pungent smell in condiments, which can also be used as an exogenetic marker for kitchen waste oil. Development of immunoassay for CPCs remains a challenging due to relatively difficult preparation of the broad-spectrum antibody (Ab). In this work, a broad-spectrum polyclonal antibody (pAb) which can simultaneously recognize capsaicin (CPC), dihydrocapsaicin (DCPC), nordihydrocapsaicin (NDCPC), and N-vanillylnonanamide (N-V) is produced, and a non-enzyme immunoassay (NISA) based on this Ab, dendritic mesoporous silica nanomaterials (DMSNs), polydopamine (PDA), and high catalytic efficiency of Pt nanoparticles to prepare signal probe (DMSNs@PDA@Pt) is established. Here, the limit of detection (LOD) of NISA for CPC is as low as 0.04 µg L-1. It is worth mentioning that the LOD of the proposed NISA is at least 23 times lower than that of traditional enzyme-linked immunosorbent assay (ELISA) based on horseradish peroxidase (HRP). Moreover, the proposed NISA is applied to detect CPCs in edible oil samples, the result has good consistency with that of ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The proposed NISA based on DMSN@PDA@Pt and broad-spectrum Ab is an ideal tool for highly effective screening CPCs for kitchen waste oil abuse surveillance.

Colorimetric and photothermal dual-mode immunosensor based on Ti3C2Tx/AuNPs nanocomposite with enhanced peroxidase-like activity for ultrasensitive detection of zearalenone in cereals.

A novel peroxidase-like nanozyme has been constructed by decorating two-dimensional Ti3C2Tx nanosheets (Ti3C2Tx NSs) with gold nanoparticles (AuNPs) to develop a colorimetric and photothermal dual-mode immunosensor. The Ti3C2Tx/AuNPs nanocomposite-catalyzed 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 reaction system produces the one-electron oxidation product of TMB (oxTMB), which exhibits color change and strong near-infrared (NIR) laser-driven photothermal effect at 808 nm laser irradiation. Given these characteristics, the developed immunosensor achieves ultrasensitive dual-mode detection of zearalenone (ZEN) by measuring colorimetric and photothermal signals with a microplate reader and a portable infrared thermometer, respectively. Under optimal working conditions, the limit of detection (LOD) of ZEN is 0.15 pg mL-1 for the colorimetric mode and 0.48 pg mL-1 for the photothermal mode. In the analysis of actual contaminated cereals samples, the test result of this method was consistent with that of UPLC-MS/MS. The proposed colorimetric and photothermal dual-mode immunosensor offers a new strategy for the low-cost detection of hazardous substances. The application of a widely used household infrared thermometer makes the signal readout more convenient, which provides great prospects in food safety and environment inspection applications.

Fluorescence immunoassay for simultaneous detection typical ß-agonists in animal derived food using blue-green upconversion nanoparticles as labels.

Common typical ß-agonists mainly include ractopamine (RAC), salbutamol (SAL), and clenbuterol (CLB). In view of the harm to human health causes by the ingestion of animal derived food containing ß-agonists, and a series of regulations have been issued to restrict the usage of ß-agonists as growth promoters. In this work, a fluorescence immunoassay is developed for the simultaneous detection of typical ß-agonists based on blue-green upconversion nanoparticles (UCNPs) combine with magnetic separation. Here, blue-green UCNPs act as a signal amplification source, and magnetic polystyrene microspheres (MPMs) act as an ideal separation medium. Based on a competitive form, capture probe competes (RAC-OVA@MPMs and SAL-OVA@MPMs) with targets to bind corresponding signal probe (anti-RAC antibody@NaYF4:Yb, Tm UCNPs and anti-SAL antibody@NaYF4:Yb, Er UCNPs). The fluorescence difference values of the competitive immune-complex obtained via magnetic separation at 483 nm and 550 nm are proportional to concentrations of RAC and SAL, respectively. The immunoassay has the wide detection linear range from 0.001 to 100 µg L-1, and the low limit of detection (LOD) is 5.04 × 10-4 µg L-1 for RAC, 1.97 × 10-4 µg L-1 for SAL, respectively. Meanwhile, use of antibody with same recognition ability for SAL and CLB makes that the fluorescence immunoassay can achieve simultaneous detection of three typical ß-agonists (RAC, SAL, and CLB). This fluorescence immunoassay has good application value and practicability for simultaneous detection of typical ß-agonists in animal derived food.

Intracellular immunization against HIV infection with an intracellular antibody that mimics HIV integrase binding to the cellular LEDGF protein.

Preventing the protein-protein interaction of the cellular chromatin binding protein Lens Epithelium-Derived Growth Factor (LEDGF) and human immunodeficiency virus (HIV) integrase is an important possible strategy for anti-viral treatment for AIDS. We have used Intracellular Antibody Capture technology to isolate a single VH antibody domain that binds to LEDGF. The crystal structure of the LEDGF-VH complex reveals that the single domain antibody mimics the effect of binding of HIV integrase to LEDGF which is crucial for HIV propagation. CD4-expressing T cell lines were constructed to constitutively express the LEDGF-binding VH and these cells showed interference with HIV viral replication, assayed by virus capsid protein p24 production. Therefore, pre-conditioning cells to express antibody fragments confers effective intracellular immunization for preventing chronic viral replication and can be a way to prevent HIV spread in infected patients. This raises the prospect that intracellular immunization strategies that focus on cellular components of viral integrase protein interactions can be used to combat the problems associated with latent HIV virus re-emergence in patients. New genome editing development, such as using CRISPR/cas9, offer the prospect intracellularly immunized T cells in HIV+ patients.

Snapin promotes HIV-1 transmission from dendritic cells by dampening TLR8 signaling.

HIV-1 traffics through dendritic cells (DCs) en route to establishing a productive infection in T lymphocytes but fails to induce an innate immune response. Within DC endosomes, HIV-1 somehow evades detection by the pattern-recognition receptor (PRR) Toll-like receptor 8 (TLR8). Using a phosphoproteomic approach, we identified a robust and diverse signaling cascade triggered by HIV-1 upon entry into human DCs. A secondary siRNA screen of the identified signaling factors revealed several new mediators of HIV-1 trans-infection of CD4+ T cells in DCs, including the dynein motor protein Snapin. Inhibition of Snapin enhanced localization of HIV-1 with TLR8+ early endosomes, triggered a pro-inflammatory response, and inhibited trans-infection of CD4+ T cells. Snapin inhibited TLR8 signaling in the absence of HIV-1 and is a general regulator of endosomal maturation. Thus, we identify a new mechanism of innate immune sensing by TLR8 in DCs, which is exploited by HIV-1 to promote transmission.

Corrigendum: CD90(+) Stromal Cells are Non-Professional Innate Immune Effectors of the Human Colonic Mucosa.

[This corrects the article on p. 307 in vol. 4, PMID: 24137162.].

Anti-melanoma activity of T cells redirected with a TCR-like chimeric antigen receptor.

Genetically modified T cells to recognize tumor-associated antigens by transgenic TCRs or chimeric antigen receptors (CAR) have been successfully applied in clinical trials. However, the disadvantages of either TCR mismatching or the requirement of a surface tumor antigen limit their wider applications in adoptive T cell therapy. A TCR-like chimeric receptor, specific for the melanoma-related gp100/HLA-A2 complex was created by joining a TCR-like antibody GPA7 with the endodomains of CD28 and CD3-ζ chain. This TCR-like CAR, GPA7-28z, was subsequently introduced into human T cells. Retargeted T cells expressing GPA7-28z could exhibit efficient cytotoxic activities against human melanoma cells in vitro in the context with HLA-A2. Furthermore, infusion of GPA7-28z-transduced T cells suppressed melanoma progression in a xenograft mouse model. Redirecting human T cells with TCR-like CARs would be a promising alternative approach to TCR-mediated therapy for melanoma patients, which is also feasible for targeting a variety of other tumor antigens.

Retargeting NK-92 for anti-melanoma activity by a TCR-like single-domain antibody.

The efficacy of immunotherapy based on natural killer (NK) cells is hampered by intrinsic non-specific cytotoxicity and insufficient activation of NK cells. Here, we confer the T-cell receptor-like (TCR-like) specificity on NK cells, taking advantage of both the innate and adaptive immune arms of the immune response to generate enhanced anti-melanoma activity. The TCR-like antibody (Ab) GPA7 was selected against melanoma-associated gp100/human leukocyte antigen (HLA)-A2 complex and then fused to intracellular domain of CD3-ζ chain. This fusion construct was incorporated into NK-92MI cell line and expressed as a chimeric antigen receptor on the surface of the cell. The anti-tumour activity of the transgenic NK-92MI-GPA7-ζ cell line was assessed against melanoma in vitro and in vivo. The engineered NK-92MI-GPA7-ζ cells could recognize melanoma cells in the context of HLA-A2 and showed enhanced killing of both melanoma cell lines and primary melanoma. Furthermore, adoptively transferred NK-92MI-GPA7-ζ cells significantly suppressed the growth of human melanoma in a xenograft model in mice. Collectively, these results demonstrate that the TCR-like Ab, GPA7, could redirect NK cells to target the intracellular antigen gp100 and enhance anti-melanoma activity, providing a promising immunotherapeutic strategy to prevent and treat melanoma.

CD90(+) Stromal Cells are Non-Professional Innate Immune Effectors of the Human Colonic Mucosa.

Immune responses at the intestinal mucosa must allow for host protection whilst simultaneously avoiding inappropriate inflammation. Although much work has focused on the innate immune functionality of hematopoietic immune cells, non-hematopoietic cell populations - including epithelial and stromal cells - are now recognized as playing a key role in innate defense at this site. In this study we examined the innate immune capacity of primary human intestinal stromal cells (iSCs). CD90(+) iSCs isolated from human colonic mucosa expressed a wide array of innate immune receptors and functionally responded to stimulation with bacterial ligands. iSCs also sensed infection with live Salmonella typhimurium, rapidly expressing IL-1 family cytokines via a RIPK2/p38MAPK-dependent signaling process. In addition to responding to innate immune triggers, primary iSCs exhibited a capacity for bacterial uptake, phagocytosis, and antigen processing, although to a lesser extent than professional APCs. Thus CD90(+) iSCs represent an abundant population of "non-professional" innate immune effector cells of the human colonic mucosa and likely play an important adjunctive role in host defense and immune regulation at this site.

The intracellular sensor NOD2 induces microRNA-29 expression in human dendritic cells to limit IL-23 release.

NOD2 is an intracellular sensor that contributes to immune defense and inflammation. Here we investigated whether NOD2 mediates its effects through control of microRNAs (miRNAs). miR-29 expression was upregulated in human dendritic cells (DCs) in response to NOD2 signals, and miR-29 regulated the expression of multiple immune mediators. In particular, miR-29 downregulated interleukin-23 (IL-23) by targeting IL-12p40 directly and IL-23p19 indirectly, likely via reduction of ATF2. DSS-induced colitis was worse in miR-29-deficient mice and was associated with elevated IL-23 and T helper 17 signature cytokines in the intestinal mucosa. Crohn's disease (CD) patient DCs expressing NOD2 polymorphisms failed to induce miR-29 upon pattern recognition receptor stimulation and showed enhanced release of IL-12p40 on exposure to adherent invasive E. coli. Therefore, we suggest that loss of miR-29-mediated immunoregulation in CD DCs might contribute to elevated IL-23 in this disease.

Conserved epitopes dominate cross-CD8+ T-cell responses against influenza A H1N1 virus among Asian populations.

Novel strains of influenza A viruses (IAVs) have emerged with high infectivity and/or pathogenicity in recent years, causing worldwide concern. T cells are correlated with protection in humans through cross-reactive immunity against heterosubtypes of IAV. However, the different hierarchical roles of IAV-derived epitopes with distinct levels of polymorphism in the cross-reactive T-cell responses against IAV remain elusive. In this study, immunodominant epitopes scattered throughout the entire proteome of 2009 pandemic influenza A H1N1 virus and seasonal IAVs were synthesized and divided into different pools depending on their conservation. The overall profile of the IAV-specific CD8(+) T-cell immunity was detected by utilizing these peptide pools and also individual peptides. A dominant role of the conserved peptide-specific T-cell immunity was illuminated within the anti-IAV responses, while the CD8(+) T-cell responses against the variable epitopes were lower than the conserved peptides. As previously demonstrated within a Caucasian population, we determined that GL9-specific T cells, which also utilize Vß 17 TCR (BV19), play a pivotal role in IAV-specific T-cell immunity within an HLA-A2(+) Asian population. Our study objectively reveals the different predominant roles of T-cell epitopes among IAV-specific cross-reactive cellular immunity. This may guide the development of vaccines with cross-T-cell immunogenicity against heterosubtypes of IAV.

Generation of single-domain antibody multimers with three different self-associating peptides.

Conventional Y-shaped antibodies have been widely used in research, diagnostics and therapeutics. Their large size result in disadvantages in certain applications, which makes production difficult. Different parts of an antibody have been used to replace the whole antibody to make it smaller. Single-domain antibodies (sdAbs) derived from the camelid heavy chain antibodies are among the smallest antibody fragments engineered for various applications. To improve the affinity of these single- sdAbs for correspondent antigens and provide suitable size of reagents for various applications, we fused an anti-epidermal growth factor receptor sdAb EG2 to self-associating peptides, RHCC derived from a right-handed coiled-coil peptide of an archaebacterium, COMPcc from human cartilage oligomeric matrix protein and C4bpα derived from human plasma C4-binding protein α-chain, respectively, to make multimeric antibodies. The multimeric antibodies were expressed as soluble cytoplasmic proteins to spontaneously form tetramers, pentamers or heptamers and were purified by affinity chromatography. The avidity of multimeric forms of sdAbs compared with that of the monomeric form of sdAbs was increased without altering binding specificity.

Weak binder for MHC molecule is a potent Mycobacterium tuberculosis-specific CTL epitope in the context of HLA-A24 allele.

Tuberculosis causes serious health problem for the world population. Antigenic peptides selected by pathogen-specific cytotoxic T lymphocytes (CTLs) are presented by major histocompatibility complex (MHC; or human leukocyte antigen [HLA] in humans) molecules, and HLA-A restricted responses may be of interest for vaccine development and the understanding of cellular immunity. A series of peptides derived from the 10-KDa culture filtrate protein (CFP10) and the 6 kDa early secretory antigenic target (ESAT-6) in the Mycobacterium tuberculosis (Mtb) have been screened and a CTL epitope restricted by the human leukocyte antigen HLA-A24, a common HLA allele in Asian people, has been identified. In this study, we studied a panel of CFP10 and ESAT-6-derived peptides to identify those with binding motifs for HLA-A24 molecules. The antigenicity of candidate peptides was assessed with in vitro refolding tests and an enzyme-linked immunospot (ELISPOT) assay, and by tetramer staining to determine the capacity to stimulate CTLs from peripheral blood mononuclear cells (PBMCs) of HLA-A24-positive TB Patients. We report that one novel candidate peptide at positions 5-14 of ESAT-6 of Mtb could induce peptide-specific CTLs from PBMCs of HLA-A24-positive patients, but not from HLA-A24-negative patients and HLA-A24-positive healthy controls. Identified epitope is a weak binder for HLA-A24 molecule in a mini MHC refolding assay. Since the peptide is presented by a common HLA class I molecule, it may be useful for immunotherapy against Mtb infection and vaccine development in the large population of Mtb-infected patients.

COMBODY: one-domain antibody multimer with improved avidity.

Antibodies (Abs) have been engineered into small antigen-binding fragments and rebuilt into multivalent high-avidity molecules for improving in vivo pharmacokinetics and efficacy in clinical use. To increase the avidity of a T-cell receptor-like single-domain Ab (sdAb) specific for HLA-A2 complex, we fused the sdAb to a coiled-coil peptide derived from human cartilage oligomeric matrix protein (COMP48) to make an sdAb multimer, termed combody. The combody improved the binding avidity of sdAb significantly, whereas the specificity for the targeted cells was retained. The strategy was also expanded to create a bispecific combody by fusing an sdAb to the N-terminal and an anti-CD3 single-chain variable fragment to the C-terminal of COMP48. The dual-specific combody was able to efficiently mediate cytotoxicity against the target cells in vitro. Taken together, the strategy to make combody could be widely adopted to increase the avidity of Ab fragment for further application.

A specific cytotoxic T-lymphocyte epitope presentation system for antitumor immunity.

The magnitude of CTL-mediated immunity response is highly dependent on the density of antigenic peptide-MHC I complexes at the cell surface. In this study, we adopt a novel strategy to promote the surface level of specific peptide-MHC I complexes. The strategy combines the inhibition of transporter associated with antigen processing (TAP) with the delivery of specific peptide into endoplasmic reticulum directly without the help of TAP. First, RNA interference (RNAi) technology was used to inhibit TAP expression for blocking endogenous epitope-assembled MHC class I on cell surface. Second, a peptide epitope of interest was covalently linked onto human beta-2-microglobulin (beta2m). Both TAP-specific siRNA and the peptide-linked beta2m were delivered into antigen-presentation cells sequentially or simultaneously using a retrovirus delivery system. The combined strategy produces a significant amount of MHC I loaded with specific epitopes on the surface while reducing endogenously peptide-assembled MHC class I both in vitro and in vivo. The efficacy of induction of specific immune response with the strategy against tumor cells is demonstrated in both tumor cell lines and a syngenic graft tumor model.

Interaction of HLA-B27 homodimers with KIR3DL1 and KIR3DL2, unlike HLA-B27 heterotrimers, is independent of the sequence of bound peptide.

HLA-B27 can form beta-2 microglobulin (beta2m)-associated heterotrimers (HLA-B27) and beta2m-free homodimers (B27(2)). Here, we study the role of complexed peptide in the interaction of these forms of B27 with the killer cell immunoglobulin (Ig)-like receptors KIR3DL1 and KIR3DL2 and with Ig-like transcripts LILRB1 and LILRB2. HLA-B27 tetramers complexed with three of five different naturally processed self peptides and three of seven pathogen-derived epitopes bound to KIR3DL1-expressing transfectants and NK cells. Heterotrimeric complexes containing peptides with charged amino acids at position 8 did not bind to KIR3DL1; however, studies with analogue peptides demonstrated that these are not the only peptide residues involved in binding. KIR3DL1 ligation by HLA-B27 inhibited NK cell IFN-gamma production in a peptide-dependent fashion. B27 but not HLA-A2, B7 or B57 heavy chains formed homodimers in the presence of peptide epitopes. B27(2) bound to KIR3DL1, KIR3DL2 and LILRB2 but not LILRB1. KIR3DL2 ligation by B27(2) inhibited NK and T cell IFN-gamma production. By contrast with HLA heterotrimers, B27(2) binding to KIR did not depend on the sequence of the bound peptide. Differences in KIR binding to classical HLA and B27(2) could be involved in the pathogenesis of spondyloarthritis.

Fully functional HLA B27-restricted CD4+ as well as CD8+ T cell responses in TCR transgenic mice.

The strong association of HLA B27 with spondyloarthropathies contrasts strikingly with most autoimmune diseases, which are HLA class II associated and thought to be mediated by CD4+ T lymphocytes. By introducing a human-derived HLA B27-restricted TCR into HLA B27 transgenic mice, we have obtained a functional TCR transgenic model, GRb, dependent on HLA B27 for response. Surprisingly, HLA B27 supported CD4+ as well as CD8+ T cell responses in vivo and in vitro. Further, HLA B27-restricted CD4+ T cells were capable of differentiation into a range of Th1 and Th2 T cell subsets with normal patterns of cytokine expression. The transgenic T cells were also able to enhance clearance of recombinant vaccinia virus containing influenza nucleoprotein in vivo. This is the first description of a human HLA class I-restricted TCR transgenic line. The existence of CD4+ MHC class I-restricted T cells has significant implications for immune regulation in autoimmunity and, in particular, in HLA B27-associated arthritis. We believe that this model provides a novel system for the study of unusual T cell behavior in vivo.

Cell-surface expression and immune receptor recognition of HLA-B27 homodimers.

OBJECTIVE: HLA-B27 is capable of forming in vitro a heavy-chain homodimer structure lacking beta(2)-microglobulin. We undertook this study to ascertain if patients with spondylarthritis express beta(2)-microglobulin-free HLA-B27 heavy chains in the form of homodimers and receptors for HLA-B27 homodimers. METHODS: Expression of HLA-B27 heavy chains by mononuclear cells was analyzed by fluorescence-activated cell sorter staining, Western blotting with the monoclonal antibody HC-10, and 2-dimensional isoelectric focusing. Fluorescence-labeled tetrameric complexes of HLA-B27 heavy-chain homodimers were constructed in which each dimer comprised one His-tagged heavy chain and one biotinylated heavy chain, and were used to stain patient and control mononuclear cells and transfected cell lines. RESULTS: Patients with spondylarthritis expressed cell-surface HLA-B27 homodimers. Populations of synovial and peripheral blood monocytes, and B and T lymphocytes from patients with spondylarthritis, and controls carried receptors for HLA-B27 homodimers. Experiments with transfected cell lines demonstrated that KIR3DL1 and KIR3DL2, and immunoglobulin-like transcript 4 (ILT4), but not ILT2, are receptors for HLA-B27 homodimers. CONCLUSION: Patients with spondylarthritis express both HLA-B27 heavy-chain homodimers and receptors for HLA-B27 homodimers. This may be of significance with regard to disease pathogenesis.