Design of a peptibody consisting of the antimicrobial peptide dhvar5 and a llama variable heavy-chain antibody fragment.

Immunoconjugates have been widely studied as potential therapeutics for infectious diseases to direct unspecific antimicrobials to pathogens. In this study, the recombinant approach was used for expression of the immunoconjugate composed of the variable domain of a llama heavy-chain antibody (VHH) against Streptococcus mutans and dhvar5, a synthetic antimicrobial peptide. Before cloning, the impact of the elongation of the peptide termini on its biological activity was evaluated by chemical synthesis of the N- or C-termini extended dhvar5 peptides. As the elongation of the C-terminus had a greater influence on decline of the antimicrobial activity, the N-terminal fusion was designed. To promote in vivo release of the active peptide, a factor Xa cleavage site was inserted between VHH and dhvar5. Propagation of transformed Escherichia coli with the constructed plasmid was only possible in the absence of isopropyl beta-D-thiogalactoside (IPTG). Although these data demonstrate that the diminished antimicrobial activity of dhvar5 by the N-terminal fusion to VHH was not sufficient for the protection of the bacterial host cells against the peptide lethal effect, an insight into propeptides biological activities may be beneficial not only for new and more successful rearrangement of the VHH-dhvar5 immunoconjugate construct, but also design of the other recombinant molecules composed of peptides toxic to host cells.

Bactericidal effects of a fusion protein of llama heavy-chain antibodies coupled to glucose oxidase on oral bacteria.

Enzymes such as lactoperoxidase and glucose oxidase (GOx) are used as antimicrobial agents in oral care products. Their low specificities and substantiveness can be reduced by covalent coupling of antimicrobial molecules to antibodies. Variable domains (V(HH)) derived from llama heavy-chain antibodies are particularly suited for such an approach. The antibodies are composed solely of heavy-chain dimers; therefore, production of active fusion proteins by using molecular biology-based techniques is less complicated than production by use of conventional antibodies. In this study, a fusion protein consisting of V(HH) and GOx was constructed and expressed by Saccharomyces cerevisiae. A llama was immunized with Streptococcus mutans strain HG982. Subsequently, B lymphocytes were isolated and cDNA fragments encoding the V(HH) fragments were obtained by reverse transcription-PCR. After construction of a V(HH) library in Escherichia coli and screening of the library against mutans group streptococci and Streptococcus sanguinis strains, we found two V(HH) fragments with high specificities for S. mutans strains. A GOx gene was linked to the two V(HH) genes and cloned into S. cerevisiae yeasts. The yeasts expressed and secreted the recombinant proteins into the growth medium. The test of binding of fusion proteins to oral bacteria through their V(HH) fragments showed that S. mutans had been specifically targeted by GOx-S120, one of the fusion protein constructs. A low concentration of the fusion protein was also able to selectively kill S. mutans within 20 min in the presence of lactoperoxidase and potassium iodide. These findings demonstrate that the fusion protein GOx-V(HH) is potentially valuable in the selective killing of target bacteria such as S. mutans.

Lactoferrin regulates proliferative response of human peripheral blood mononuclear cells to phytohemagglutinin and mixed lymphocyte reaction.

The aim of this study was to investigate the effects of lactoferrin (LF) on the proliferative response of human peripheral blood mononuclear cells (PBMC) induced by phytohemagglutinin (PHA) and alloantigens in a two-way mixed lymphocyte reaction (MLR). The proliferative responses were measured by MTT colorimetric assay and presented as a proliferation index (PI) or as changes in PI caused by LF relative to PHA or MLR controls. We found that the effects of LF in both experimental models were differential and dependent on an individual PBMC reactivity, mitogen or alloantigen and LF concentration. Generally, lymphocytes from donors responsive to LF exhibited higher proliferation indices to PHA when compared with non-responsive individuals. Lactoferrin at low doses showed regulatory effects, whereas at higher doses the proliferation was significantly reduced. Mixed lymphocyte reaction was generally inhibited by LF. The results suggest that the differential action of LF might be due to its ability to sense the activation status of lymphocytes.