NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/VHH) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/VHH from a humanized-camel VH/VHH display library. VH/VHH from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3′di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed VHH hallmark and were designated VHH6 and VHH24; other clones were conventional VH, designated VH9 and VH13. All VH/VHH were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, VHH6 and VHH24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/VHH mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.

Although the importance of the macrophage complement receptor immunoglobulin (CRIg) in the phagocytosis of complement opsonized bacteria and in inflammation has been established, the regulation of CRIg expression remains undefined. Because cellular activation during inflammation leads to the release of arachidonate, a stimulator of leukocyte function, we sought to determine whether arachidonate regulates CRIg expression. Adding arachidonate to maturing human macrophages and to prematured CRIg ؉ macrophages caused a significant decrease in the expression of cell-surface CRIg and CRIg mRNA. This effect was independent of the metabolism of arachidonate via the cyclooxygenase and lipoxygenase pathways, because it was not inhibited by the nonsteroidal anti-inflammatory drugs indomethacin and nordihydroguaiaretic acid. Studies with specific pharmacological inhibitors of arachidonate-mediated signaling pathways showed that protein kinase C was involved. Administration of dexamethasone to macrophages caused an increase in CRIg expression. Studies with proinflammatory and immunosuppressive cytokines showed that IL-10 increased, but interferon-␥, IL-4, and transforming growth factor-␤1 decreased CRIg expression on macrophages. This down-and upregulation of CRIg expression was reflected in a decrease and increase, respectively, in the phagocytosis of complement opsonized Candida albicans. These data suggest that a unique inflammatory mediator network regulates CRIg expression and point to a mechanism by which arachidonate and dexamethasone have reciprocal effects on inflammation.

A cell penetrating format of human single chain antibody (HuScFv) specific to matrix protein (M1) of influenza A virus was produced by molecular linking of the gene sequence encoding the HuScFv (huscfv) to a protein transduction domain, i.e., penetratin (PEN) of the Drosophila homeodomain. DNA of a recombinant phagemid vector carrying the huscfv was used as a platform template in a three-step PCR for generating a nucleotide sequence encoding a 16 amino acid PEN peptide. The PEN-HuScFv had negligible cytotoxicity on living MDCK cells. They were readily translocated across the cell membrane and bound to native M1 in the A/H5N1-infected cells as revealed by immunofluorescent confocal microscopy. The PEN-HuScFv, when used to treat the influenza virus infected cells, reduced the number of viruses released from the cells. In conclusion, the cell penetrating M1-specific HuScFv, a transbody, produced in this study affected the influenza A virus life cycle in living mammalian cells. While the molecular mechanisms of the PEN-HuScFv need more investigation, the reagent warrants further testing in animals before developing it into a human immunotherapeutic anti-influenza formula.