Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics.

Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance. In this study, we have further tailored the design of human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the Dock'n'Flash technology. In more detail, we have generated the 2nd generation of scFvs carrying the photoreactive UAA p-benzoyl-l-phenylalanine (pBpa). Based on key properties, such as expression levels, activity, and affinity, a preferred choice of site for pBpa, located in the beginning of the C-terminal affinity-tag, was for the first time pin-pointed. Further, the results showed that pBpa mutated antibody could be site-specifically photocoupled to free and surface immobilized ß-cyclodextrin (an affinity ligand to pBpa). This paves the way for use of scFv antibodies, engineered for site-specific photochemical-based tagging, functionalization, and orientated surface immobilization, in affinity proteomics.

Broad-Spectrum Antimicrobial Star Polycarbonates Functionalized with Mannose for Targeting Bacteria Residing inside Immune Cells.

In this study, a series of star-shaped polycarbonates are synthesized by metal-free organocatalytic ring-opening polymerization of benzyl chloride (BnCl) and mannose-functionalized cyclic carbonate monomers (MTC-BnCl and MTC-ipman) with heptakis-(2,3-di-O-acetyl)-ß-cyclodextrin (DA-ß-CD) as macroinitiator. The distributions and compositions of pendent benzyl chloride and protected mannose group (ipman) units are facilely modulated by varying the polymerization sequence and feed ratio of the monomers, allowing precise control over the molecular composition, and the resulting polymers have narrow molecular weight distribution. After deprotection of ipman groups and quaternization with various N,N-dimethylalkylamines, these star polymers with optimized compositions of cationic and mannose groups in block and random forms exhibit strong bactericidal activity and low hemolysis. Furthermore, the optimal mannose-functionalized polymer demonstrates mannose receptor-mediated intracellular bactericidal activity against BCG mycobacteria without inducing cytotoxicity on mammalian cells at the effective dose. Taken together, the materials designed in this study have potential use as antimicrobial agents against diseases such as tuberculosis, which is caused by intracellular bacteria.

Hydroxypropyl-ß-cyclodextrin spikes local inflammation that induces Th2 cell and T follicular helper cell responses to the coadministered antigen.

Cyclodextrins are commonly used as a safe excipient to enhance the solubility and bioavailability of hydrophobic pharmaceutical agents. Their efficacies and mechanisms as drug-delivery systems have been investigated for decades, but their immunological properties have not been examined. In this study, we reprofiled hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as a vaccine adjuvant and found that it acts as a potent and unique adjuvant. HP-ß-CD triggered the innate immune response at the injection site, was trapped by MARCO(+) macrophages, increased Ag uptake by dendritic cells, and facilitated the generation of T follicular helper cells in the draining lymph nodes. It significantly enhanced Ag-specific Th2 and IgG Ab responses as potently as did the conventional adjuvant, aluminum salt (alum), whereas its ability to induce Ag-specific IgE was less than that of alum. At the injection site, HP-ß-CD induced the temporary release of host dsDNA, a damage-associated molecular pattern. DNase-treated mice, MyD88-deficient mice, and TBK1-deficient mice showed significantly reduced Ab responses after immunization with this adjuvant. Finally, we demonstrated that HP-ß-CD-adjuvanted influenza hemagglutinin split vaccine protected against a lethal challenge with a clinically isolated pandemic H1N1 influenza virus, and the adjuvant effect of HP-ß-CD was demonstrated in cynomolgus macaques. Our results suggest that HP-ß-CD acts as a potent MyD88- and TBK1-dependent T follicular helper cell adjuvant and is readily applicable to various vaccines.

Optimization of the entrapment of bacterial cell envelope extracts into microparticles for vaccine delivery.

The encapsulation of a Brucella ovis extract (HS) in microparticles has been proved effective against experimental infections in mice. This work describes different strategies to increase the HS loading and prepare large batches as necessary to test this vaccine in ovine. The mixture of HS with beta-cyclodextrin was optimized in order to increase the HS loading in microparticles. On the other hand, TROMS ('Total Recirculation One-Machine System') led microparticles with a more homogeneous size than the laboratory or standard procedure. Moreover, the initial burst release of HS from the standard microparticles was higher than for the TROMS ones. In fact, standard microparticles displayed a higher amount of adsorbed HS. On the contrary, both preparative methods were found effective to preserve the integrity and anti-genicity of the loaded HS. In summary, beta-CD can be used to increase the loading of large hydrophobic materials and TROMS is a valid large production of antigen-loaded microparticles.