Group B Streptococcus chimeric capsular polysaccharides as novel multivalent vaccine candidates.

The capsular polysaccharide of the human pathogen Group B Streptococcus is a key virulence factor and vaccine candidate that induces protective antibodies when conjugated to carrier proteins. It consists of long polymeric chains of oligosaccharide repeating units, and each of the ten capsular serotypes described so far presents a unique chemical structure with distinct antigenic properties; therefore, broad protection against this pathogen could be achieved by a combination of ten glycoconjugates. Capsular polysaccharide biosynthesis and assembly follow a polymerase-dependent pathway that is widespread in encapsulated bacteria and is encoded by a polycistronic operon. Here we exploited the sequence similarity between the capsule operons of types V and IX to generate hybrid polysaccharides incorporating epitopes of both serotypes in a single molecule, by co-expressing their specific CpsM, O, I glycosyltransferases in a single isolate. Physicochemical and immunochemical methods confirmed that an engineered strain produced a high molecular weight chimeric polysaccharide, combining antigenic specificities of both type V and IX. By optimizing the copy number of key glycosyltransferase genes, we were able to modulate the ratio between type-specific epitopes. Finally, vaccination with chimeric glycoconjugates significantly decreased the incidence of disease in pups born from immunized mice challenged with either serotype. This study provides proof of concept for a new generation of glycoconjugate vaccines that combine the antigenic specificity of different polysaccharide variants in a single molecule, eliciting a protective immune response against multiple serotype variants.

Chemical Conjugate of Low Molecular Weight Heparin and Suramin Fragment Inhibits Tumor Growth Possibly by Blocking VEGF165.

Low molecular weight heparin (LMWH) and its derivatives have been reported to possess antiangiogenic effect via electrostatic interaction with various angiogenic growth factors such as VEGF165. However, clinical applications of LMWH for anticancer therapy have been restricted due to its anticoagulant effect and insufficient therapeutic efficacy. To overcome these limitations and enhance the antiangiogenic efficacy, LMWH was conjugated with suramin fragments that have a binding affinity to the heparin-binding domain (HBD) of proteins. The conjugation of suramin fragments to LMWH enhanced the antiangiogenic effect of LMWH by increasing the binding affinity to VEGF165, while decreasing its anticoagulant activity. The chemical conjugate of LMWH and suramin fragments (LHsura) showed a substantial inhibitory effect on VEGF165-mediated cell proliferation, migration, and tube formation of HUVECs without significant cytotoxicity in vitro. Finally, we confirmed the anticancer effect of LHsura (61.4% vs control) in a SCC7-bearing mouse model.

Novel Chitosan-Riboflavin Conjugate with Visible Light-Enhanced Antifungal Properties against Penicillium digitatum.

A novel chemical conjugate between chitosan (CH) and riboflavin (RF) has been synthesized and characterized via Fourier transform infrared, NMR, and other spectroscopic methods. Photophysical and photochemical properties such as absorption spectra, fluorescence emission, fluorescence anisotropy, and singlet oxygen generation were characterized as well. This new biopolymer-based conjugate was designed to have an antifungal effect enhanced through antimicrobial photodynamic therapy. The antifungal effect of this conjugate (CH-RF) was compared with CH and RF against Penicillium digitatum in vitro. The conjugate showed the highest fungal growth inhibition of all systems tested at a dose of 0.5% w/v. This new biopolymer-based compound could be a promising alternative to fungicides used in citrus fruits postharvest.

Design and Synthesis of a Peptide-Based Glioma-Targeted Drug Delivery Vector gHope2.

In the current chapter, we are detailing the synthesis path of a tumor-targeted, CPP-functionalized chemotherapeutic drug, as well as in vitro validation of the targeting and cell penetrating functionalities of the construct. The design of targeted drug delivery vehicle is based on a new glioma-specific homing peptide that has been conjugated to doxorubicin. Further functionalization with an 18-amino acid cell penetrating peptide pVEC was achieved, a CPP that was chosen because of its high cell penetrating efficacy and low toxicity. The three elements were combined into one drug delivery construct gHope2, and its tumor-homing and cell penetrating activity was demonstrated in human glioma cell line U87.

Immunogenicity characterization of genetically fused or chemically conjugated heat-stable toxin toxoids of enterotoxigenic Escherichia coli in mice and pigs.

Enterotoxigenic Escherichia coli (ETEC) producing type Ib heat-stable toxin (STa) are a main cause of children's diarrhea and travelers' diarrhea, thus STa needs to be targeted in ETEC vaccine development. However, because this 19-amino acid STa is poorly immunogenic, attempts to genetically fuse or chemically couple it to carrier proteins have been made to enhance STa immunogenicity. In this study, we selected one genetic fusion and one chemical conjugate to comparatively evaluate STa immunogenicity. The genetic fusion is 3xSTaN12S-mnLTR192G/L211A carrying three toxoid (STaN12S) genetically fused to a double mutant LT monomer (mnLTR192G/L211A); the chemical conjugate is BSA-STaA14T, which has toxoid STaA14T chemically coupled to bovine serum albumin (BSA). We immunized mice with the STa toxoid fusion and chemical conjugates, and examined antibody responses. Furthermore, we immunized pigs and evaluated derived antibodies for efficacy to passively provide protection against ETEC diarrhea using a piglet model. Data showed that mice subcutaneously immunized with BSA-STaA14T or 3xSTaN12S-mnLTR192G/L211A developed a strong anti-STa antibody, and the induced antibodies exhibited equivalent toxin-neutralizing activities. Pigs immunized with 3xSTaN12S-mnLTR192G/L211A or BSA-STaA14T developed similar levels of anti-STa antibodies; piglets with passively acquired antibodies induced by the genetic fusion appeared better protected against STa + ETEC. Results from the current study indicate that the fusion and conjugate approaches are viable options for facilitating STa immunogenicity and developing ETEC vaccines.

Confirmation of covalently-linked structure and cell-death inducing activity in site-specific chemical conjugates of human Fas ligand extracellular domain.

OBJECTIVE: In this study, we aimed to identify the structural components and to clarify the biological activity in the site-specific conjugates of human Fas ligand extracellular domain (hFasLECD) with either fluorescein moiety (FL) or chicken egg-white avidin (Avi). The conjugates were characterized by molecular-weight measurement using MALDI-TOF mass-spectrometric analysis and by cell-death inducing activity measurement against a human colorectal cancer cell line, HT-29, using MTT cell-viability assay. Pretreatment effect with human interferon-γ (IFN-γ) on the cell-death inducing activity was evaluated. RESULTS: The mass-spectrometric analysis of the hFasLECD-Avi conjugate showed that it was possible to detect the signal peak of molecular-weight to electric charge (m/z) derived from the component involved in the covalent linking as the sum of the molecular-weight of unconjugated hFasLECD- and Avi-derivative subunits, in addition to the signals from each corresponding subunit component irrelevant to the covalent linking. The cell-viability assay revealed that both conjugates possessed a remarkable death-inducing activity against HT-29 cells in synergy with the pretreatment using human IFN-γ. Following 24 h pretreatment with 100 IU/ml of human IFN-γ, almost no viable cells existed after 72 h treatment with either 100 or 1000 ng/ml of FL-hFasLECD and hFasLECD-Avi conjugates.

High-Yield Synthesis of Monomeric LMWP(CPP)-siRNA Covalent Conjugate for Effective Cytosolic Delivery of siRNA.

Because of the unparalleled efficiency and universal utility in treating a variety of disease types, siRNA agents have evolved as the future drug-of-choice. Yet, the inability of the polyanionic siRNA macromolecules to cross the cell membrane remains as the bottleneck of possible clinical applications. With the cell penetrating peptides (CPP) being discovered lately, the most effective tactic to achieve the highest intracellular siRNA delivery deems to be by covalently conjugating the drug to a CPP; for instance, the arginine-rich Tat or low molecular weight protamine (LMWP) peptides. However, construction of such a chemical conjugate has been referred by scientists in this field as the "Holy Grail" challenge due to self-assembly of the cationic CPP and anionic siRNA into insoluble aggregates that are deprived of the biological functions of both compounds. Based on the dynamic motion of PEG, we present herein a concise coupling strategy that is capable of permitting a high-yield synthesis of the cell-permeable, cytosol-dissociable LMWP-siRNA covalent conjugates. Cell culture assessment demonstrates that this chemical conjugate yields by far the most effective intracellular siRNA delivery and its corresponded gene-silencing activities. This work may offer a breakthrough advance towards realizing the clinical potential of all siRNA therapeutics and, presumably, most anionic macromolecular drugs such as anti-sense oligonucleotides, gene compounds, DNA vectors and proteins where conjugation with the CPP encounters with problems of aggregation and precipitation. To this end, the impact of this coupling technique is significant, far-reaching and wide-spread.

Novel hyaluronic acid-methotrexate conjugate suppresses joint inflammation in the rat knee: efficacy and safety evaluation in two rat arthritis models.

BACKGROUND: Methotrexate (MTX) is one of the most widely used medications to treat rheumatoid arthritis (RA), and recent studies have also suggested the potential benefit of the drug for the treatment of osteoarthritis (OA) of the knee. MTX is commonly administered in oral formulations, but is often associated with systemic adverse reactions. In an attempt to address this issue, we have shown previously that a conjugate of hyaluronic acid (HA) and MTX exhibits potential as a drug candidate for intra-articular treatment of inflammatory arthritis. In this study, we compare the efficacy and safety of an optimized HA-MTX conjugate, DK226, with that of MTX in inflammatory arthritis rat models. METHODS: In vitro activity of DK226 was assessed in human fibroblast-like synoviocytes (HFLS) and a synovial sarcoma cell line, SW982. Release of MTX from DK226 was investigated after incubation with rabbit synovial tissue homogenate or synovial fluid. In vivo efficacy of DK226 was evaluated in antigen-induced arthritis (AIA) and collagen-induced arthritis (CIA) in the rat knee. Pharmacokinetics and hematological toxicity after treatment with oral MTX or an intra-articular injection of DK226 were compared in AIA. RESULTS: Proliferation of HFLS and SW982 cells was inhibited by DK226, and the inhibitory activity was reversed by cotreatment with excess HA or anti-CD44 antibody. MTX was released from DK226 by incubation with rabbit synovial tissue homogenate or synovial fluid at pH 4.0, but not at pH 7.4. AIA was ameliorated by intra-articular DK226, but not by HA, as potently as oral MTX. Hematological toxicity was induced by oral MTX, but not by DK226. The maximum plasma concentration of MTX after oral MTX was 40 times higher than the concentration of MTX after an intra-articular injection of DK226. Knee swelling in AIA was inhibited by intra-articular injections of DK226, but not by free MTX or a mixture of HA and MTX. In CIA, an injection of DK226 into the right knee joint significantly reduced swelling and synovial inflammation of the treated knee joint, but had no effect on the untreated contralateral knee joint. CONCLUSIONS: DK226 exerted anti-arthritic effects in two different models of arthritis. The conjugate had a wider therapeutic window than oral MTX, and could be a future drug for treatment of arthritic disorders, including inflammatory OA.