Structural insights into antigen recognition of an anti-ß-(1,6)-ß-(1,3)-D-glucan antibody.

Schizophyllan (SCH) is a high molecular weight homopolysaccharide composed of a ß-(1,3)-D-glucan main chain with branching ß-(1,6)-bound D-glucose residues. It forms triple helices that are highly stable towards heat and extreme pH, which provides SCH with interesting properties for industrial and medical applications. The recombinant anti-SCH antibody JoJ48C11 recognizes SCH and related ß-(1,6)-branched ß-(1,3)-D-glucans, but details governing its specificity are not known. Here, we fill this gap by determining crystal structures of the antigen binding fragment (Fab) of JoJ48C11 in the apo form and in complex with the unbranched ß-(1,3)-D-glucose hexamer laminarihexaose 3.0 and 2.4 Å resolution, respectively. Together with docking studies, this allowed construction of a JoJ48C11/triple-helical SCH complex, leading to the identification of eight amino acid residues of JoJ48C11 (Tyr27H, His35H, Trp47H, Trp100H, Asp105H; Asp49L, Lys52L, Trp90L) that contribute to the recognition of glucose units from all three chains of the SCH triple helix. The importance of these amino acids was confirmed by mutagenesis and ELISA-based analysis. Our work provides an explanation for the specific recognition of triple-helical ß-(1,6)-branched ß-(1,3)-D-glucans by JoJ48C11 and provides another structure example for anti-carbohydrate antibodies.

An Antigen-Free, Plasmacytoid Dendritic Cell-Targeting Immunotherapy To Bolster Memory CD8+ T Cells in Nonhuman Primates.

The priming, boosting, and restoration of memory cytotoxic CD8+ T lymphocytes by vaccination or immunotherapy in vivo is an area of active research. Particularly, nucleic acid-based compounds have attracted attention due to their ability to elicit strong Ag-specific CTL responses as a vaccine adjuvant. Nucleic acid-based compounds have been shown to act as anticancer monotherapeutic agents even without coadministration of cancer Ag(s); however, so far they have lacked efficacy in clinical trials. We recently developed a second-generation TLR9 agonist, a humanized CpG DNA (K3) complexed with schizophyllan (SPG), K3-SPG, a nonagonistic Dectin-1 ligand. K3-SPG was previously shown to act as a potent monoimmunotherapeutic agent against established tumors in mice in vivo. In this study we extend the monoimmunotherapeutic potential of K3-SPG to a nonhuman primate model. K3-SPG activated monkey plasmacytoid dendritic cells to produce both IFN-α and IL-12/23 p40 in vitro and in vivo. A single injection s.c. or i.v. with K3-SPG significantly increased the frequencies of activated memory CD8+ T cells in circulation, including Ag-specific memory CTLs, in cynomolgus macaques. This increase did not occur in macaques injected with free CpG K3 or polyinosinic-polycytidylic acid. Injection of 2 mg K3-SPG induced mild systemic inflammation, however, levels of proinflammatory serum cytokines and circulating neutrophil influx were lower than those induced by the same dose of polyinosinic-polycytidylic acid. Therefore, even in the absence of specific Ags, we show that K3-SPG has potent Ag-specific memory CTL response-boosting capabilities, highlighting its potential as a monoimmunotherapeutic agent for chronic infectious diseases and cancer.

Complex Consisting of ß-Glucan and Antigenic Peptides with Cleavage Site for Glutathione and Aminopeptidases Induces Potent Cytotoxic T Lymphocytes.

The efficient induction of antigen-specific immune responses requires not only promotion of the uptake of antigens and adjuvant molecules into antigen-presenting cells but also control of their intracellular behavior. We previously demonstrated that the ß-glucan schizophyllan (SPG) can form complexes with CpG oligonucleotides with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses. In this study, we prepared various conjugates composed of antigenic peptide (OVA257-264) and dA40 and made complexes with SPG. The conjugates with a disulfide bond between OVA257-264 and dA40 were easily cleaved by glutathione. The resultant peptides with a hydrophobic amino acid at the C-terminal end was recognized by puromycin-insensitive leucine aminopeptidase (PILS-AP), which trims antigenic peptide precursors and prepares peptides of eight or nine amino acids in length, which is the optimal length for binding to major histocompatibility complex (MHC)-I. The conjugate exposed to such enzymes induced a high antigen presentation level. The antigen presentation level was almost the same before and after the complexation with SPG. Immunization with a mixture of dA-OVA257-264/SPG and CpG-dA/SPG induced high antigen-specific cytotoxic T-lymphocyte activity at a much lower peptide dose than in previous studies. These results can be strongly ascribed to not only the cell-specific delivery by SPG but also the control of the intracellular behavior by the introduction of cleavage sites. Therefore, peptide-dA/SPG complexes could be used as potent vaccine antigens for the treatment of cancers and infectious diseases.

Immunization with antigenic peptides complexed with ß-glucan induces potent cytotoxic T-lymphocyte activity in combination with CpG-ODNs.

The induction of antigen-specific immune responses requires immunization with not only antigens, but also adjuvants. CpG oligonucleotides (CpG-ODNs) are well-known ligands for Toll-like receptor 9 and a potent adjuvant that induces both Th1-type humoral and cellular immune responses including cytotoxic T-lymphocyte responses. We previously demonstrated that ß-glucan schizophyllan (SPG) can form complexes with CpG-ODNs with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses by co-administration of antigenic proteins, namely ovalbumin (OVA). Immunization with antigenic peptides, OVA257-264, did not induce these antigen-specific immune responses even in combination with CpG-dA/SPG, indicating that peptides require a carrier to antigen presenting cells. In this study, we prepared conjugates comprising OVA257-264 and dA40, and made complexes with SPG. Immunization with OVA257-264-dA/SPG induced peptide-specific immune responses in combination with CpG-dA regardless of complexation with SPG both in vitro and in vivo. When splenocytes from immunized mice were incubated with E.G7-OVA tumor model cells presenting OVA peptides, the number of cells drastically decreased after 24h. Furthermore, mice pre-immunized with OVA257-264-dA/SPG and CpG-ODNs exhibited a long delay in tumor growth after tumor inoculation. Therefore, these peptide-dA/SPG and CpG-dA/SPG complexes could be used as a potent vaccine for the treatment of cancers and infectious diseases.

Synthesis of the HSA-conjugate of the S-linked thiomimetic of the branched tetrasaccharide repeating unit of the immunostimulant polysaccharide, schizophyllan. Evaluation as potential immunomodulator.

The conjugate with human serum albumin (HSA) of the S-linked thioanalogue of the branched tetrasaccharide repeating unit of the polysaccharide, schizophyllan, was synthesized from 1,2,4,6-tetra-O-acetyl-3-S-[2,4-di-O-acetyl-3,6-di-S-(2,3,4,6-tetra-O-ac etyl-beta-D-glucopyranosyl)-3,6-dithio-beta-D-glucopyranosyl]-3-thio-bet a-D-glucopyranose [M.O. Contour-Galcera et al., Carbohydr. Res., 281 (1996) 119-128] in five steps, and its potential immunomodulatory activity was evaluated in human blood mononuclear cells. The protein glycoconjugate did not effect proliferation or production of IL-4, IL-5 and IFN-g in a significant way.

Correlation between immunological activity, molar mass, and molecular structure of different (1-->3)-beta-D-glucans.

(1-->3)-beta-D-Glucans are said to be potential biomedical drugs against bacterial or viral infections and also show antitumour activity. These substances seem to enhance the activity of the immune system, but today there is no accepted mechanism, and not even any agreement on the parameters which influence the activity. Therefore, glucans with different structures and/or varying molar mass were characterized by multi-angle laser-light scattering coupled with size-exclusion chromatography in order to obtain the molar mass distribution and to gain an idea of the structure in solution. After ensuring that all samples were free of pyrogens, the Tumour-necrosis-factor-alpha release activity and the superoxide-anion released from human blood monocytes were measured in groups of samples with comparable polydispersity and degree of substitution. All glucans investigated, regardless of molar mass and solution structure, stimulate the investigated immunological measures more than a commercially available biomedical drug used for comparison. The greatest magnitude of molar mass was found to be about 550,000 g/mol for all the glucans investigated. Contrary to the cited literature, helical structures were not essential, and not even advantageous, for immunological activity.

Epitope analysis using anti-oligosaccharide (G4) monoclonal antibody.

A murine monoclonal antibody recognizing (1-->6)-beta-D-glucopyranosyl laminaritriose (G4) was prepared by immunizing BALB/c mice with G4-bovine serum albumin conjugate and fusing the splenocytes with mouse myeloma cells. The monoclonal antibody (IgM) provoked by the cloned cells showed low reactivity with schizophyllan, an antitumor polysaccharide, but notable reactivity with some low-molecular-weight schizophyllans. This antibody was useful for determination of the epitope of several polysaccharides. The extent of reactivity of this monoclonal antibody was related only to the molecular weight of schizophyllan.

Detection of immunoreactive schizophyllan by solid-phase enzyme-linked immunosorbent assay.

In order to detect immunoreactive schizophyllan (SPG), one of the beta-1,3-glucans in solution, a murine anti-schizophyllan monoclonal antibody, SPG1-HS, was used as a primary antibody for solid-phase enzyme-linked immunosorbent assay (ELISA). Polysaccharide-bovine serum albumin (BSA), and polysaccharide-bovine hemoglobin (Hb) conjugate were prepared as an antigen. The absorbance at 490 nm in ELISA was directly proportional to the SPG concentration in SPG-BSA or SPG-Hb conjugates, allowing the accurate measurement of SPG at concentrations of more than 1.0 microgram/ml. Also, this ELISA detected SPG in SPG-BSA and SPG-Hb conjugates in human serum in a similar manner. These results suggest that this ELISA is applicable for the determination of SPG concentration in SPG-BSA and SPG-Hb conjugates in human tissue and blood.

Distribution of TNF endogenously induced by various immunopotentiators and Lactobacillus casei in mice.

Intravenous administration of heat-killed Lactobacillus casei YIT 9018 (LC) elicited endogenous cytotoxic factor (CF) production in ICR mice, peaking in serum after 2 h and declining gradually to basal level at 23 h. The endogenous CF production was significantly enhanced by priming with high molecular-weight lignins and glucans, but not by phenylpropenoid precursors or partially hydrolyzed products of glucans. The extent of stimulation of CF production by these priming agents was positively related to that of tumor necrosis factor (TNF) production, as judged by enzyme-linked immunosorbent assay. Endogenously produced TNF was concentrated more in liver, lung and intestine, as well as in serum, than in other organs. Histochemical examination revealed a significant increase in the number and swelling of Kupffer cells and sinusoidal endothelium in the liver of the treated mice.

Preparation of polyclonal antibodies to an anti-tumor (1----3)-beta-D-glucan, schizophyllan.

Antibodies against Schizophyllan (SPG) or SPG conjugated with bovine serum albumin (BSA) were raised in rabbits by multiple subcutaneous immunization. The titer of SPG-reactive antibodies in the antiserum to SPG-BSA conjugate was significantly higher than in the antiserum to SPG as assessed by enzyme-linked immunosorbent assay (ELISA). The SPG-reactive antibodies purified by affinity chromatography using a Protein A-Sepharose CL-4B column interacted with the SPG-BSA conjugate spotted on a nitrocellulose membrane filter in a dose-dependent manner, but the anti-SPG antibodies did not interact with BSA. Immunocytochemical staining has also shown that the anti-SPG antibodies reacted with SPG taken up by mouse peritoneal macrophages.