Bionanocapsule-based enzyme-antibody conjugates for enzyme-linked immunosorbent assay.

Macromolecules that can assemble a large number of enzyme and antibody molecules have been used frequently for improvement of sensitivities in enzyme-linked immunosorbent assays (ELISAs). We generated bionanocapsules (BNCs) of approximately 30nm displaying immunoglobulin G (IgG) Fc-binding ZZ domains derived from Staphylococcus aureus protein A (designated as ZZ-BNC). In the conventional ELISA using primary antibody and horseradish peroxidase-labeled secondary antibody for detecting antigen on the solid phase, ZZ-BNCs in the aqueous phase gave an approximately 10-fold higher signal. In Western blot analysis, the mixture of ZZ-BNCs with secondary antibody gave an approximately 50-fold higher signal than that without ZZ-BNCs. These results suggest that a large number of secondary antibody molecules are immobilized on the surface of ZZ-BNCs and attached to antigen, leading to the significant enhancement of sensitivity. In combination with the avidin-biotin complex system, biotinylated ZZ-BNCs showed more significant signal enhancement in ELISA and Western blot analysis. Thus, ZZ-BNC is expected to increase the performance of various conventional immunoassays.

Inhibition of measles virus and subacute sclerosing panencephalitis virus by RNA interference.

Subacute sclerosing panencephalitis (SSPE) is a rare, but fatal outcome of measles virus (MeV) infection. SSPE develops after prolonged persistence of mutated MeV called SSPE virus. Although a combination therapy using interferon and inosiplex or ribavirin appears to prolong survival time to some extent, there is currently no effective treatment to completely cure SSPE and a new treatment strategy is greatly needed. In this study, we adopted RNA interference (RNAi) strategy and examined whether small interfering RNAs (siRNAs) can be used to inhibit replication of MeV and SSPE virus. We report here that siRNAs targeted against L mRNA of MeV, either synthetic siRNAs or those generated by pcPUR+U6i-based expression plasmids, effectively and specifically inhibited replication of both MeV and SSPE virus without exhibiting any cytotoxic effect. The L protein of MeV is a major component of RNA-dependent RNA polymerase that is essential for viral RNA replication, and yet it is least abundant among all the MeV proteins expressed. Therefore, mRNA encoding the L protein would be a good target for RNAi strategy. The present results imply the possibility that our siRNAs against MeV L mRNA are among the potential candidates to be used to treat patients with SSPE.

Nanocapsules incorporating IgG Fc-binding domain derived from Staphylococcus aureus protein A for displaying IgGs on immunosensor chips.

To enhance the sensitivities and antigen-binding capacities of immunosensors, oriented immobilization of antibodies on the surface of the sensor chip is critical, but to date, this has not been adequately achieved. We describe a way of adsorbing immunoglobulin (Ig) proteins onto 32-nm bio-nanocapsules (BNCs) through IgG Fc-binding domains derived from Staphylococcus aureus protein A (ZZ-BNC). This arrangement permits approximately 60 molecules of mouse total IgG bind to ZZ-BNC and all the IgG Fv regions to be displayed outwardly for the effective binding of antigens. ZZ-BNCs adsorbed onto the gold surface of the sensor chip of the quartz crystal microbalance (QCM) could markedly enhance the sensitivity and antigen-binding capacity of the chip. On the sensor chip of surface plasmon resonance (SPR), antibodies on the ZZ-BNCs showed higher affinities to each antigen than those on protein A. The BNC-coated sensor chip is very stable, and should prove useful for various immunosensor applications due to oriented immobilization of antibodies.

In vivo delivery of bionanocapsules displaying Phaseolus vulgaris agglutinin-L4 isolectin to malignant tumors overexpressing N-acetylglucosaminyltransferase V.

Metastasis is a key aspect of tumor malignancy, and several malignant tumors show expression of various mature N-type glycans. In particular, beta1-6 branching N-acetylglucosamine (GlcNAc) is abundantly expressed as a part of high-mannose glycans in various highly metastatic cancers. Phaseolus vulgaris agglutinin-L(4) isolectin (L(4)-PHA), which adheres to beta1-6 GlcNAc specifically, has been used for in situ cancer diagnosis. Bionanocapsules (BNCs), hollow particles with a diameter of approximately 80 nm and composed of hepatitis B surface antigen (HBsAg) and a lipid bilayer, have been developed as human liver-specific nanocapsules for in vivo drug delivery system. In this study, we have generated L(4)-PHA-displaying BNCs (PHA-BNCs) and examined whether L(4)-PHA could retarget the BNCs to malignant tumors as a "biosensor" distinguishing tumor metastaticity. Fluorescence-labeled PHA-BNCs injected systemically into a mouse xenograft model were found to accumulate in beta1-6 GlcNAc-expressing malignant tumors. The PHA-BNCs were able to deliver DNA to the malignant cancer cells. These results open up the possibility of using L(4)-PHA lectin as a targeting molecule in a drug delivery system, and of using PHA-BNCs as a novel nanodevice for malignant tumor-specific bioimaging and drug delivery.

Nonstructural proteins 4A and 4B of hepatitis C virus transactivate the interleukin 8 promoter.

Interleukin 8 (IL-8) is induced in many cell types by various stimuli including virus infection. It was reported that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) was involved in induction of IL-8 expression at both mRNA and protein levels in cultured human cells. In this study, we aimed to determine whether or not another HCV protein(s) transactivates the IL-8 gene expression, by means of an IL-8 promoter-driven luciferase reporter assay and measurement of endogenous IL-8 mRNA and secreted IL-8 protein levels. We observed that NS4B, and NS4A to a lesser extent, significantly transactivated the IL-8 promoter, which resulted in enhanced production of IL-8 protein. Also, the IL-8 expression was augmented in Huh-7 cells harboring an HCV subgenomic RNA replicon, compared with the control cells. Deletion mutational analysis of the IL-8 promoter revealed the possible involvement of the transcription factor AP-1 in both NS4A- and NS4B-mediated IL-8 gene activation. In addition, the IL-8 gene activation by NS4B, but not that by NS4A, was likely to involve NF-kappaB and/or NFIL-6. The degree of the transactivation by NS4B and NS4A varied with different human cell lines, with HeLa cells showing the strongest activation followed by Huh-7 cells, and with HepG2 cells exhibiting a marginal level of activation. Taken together, our present results suggest the possibility that NS4B and NS4A play an important role in inducing the IL-8 gene expression under certain cellular conditions, which might be one of the strategies to establish persistent HCV infection.

Hepatitis C virus NS5A protein interacts with 2',5'-oligoadenylate synthetase and inhibits antiviral activity of IFN in an IFN sensitivity-determining region-independent manner.

The non-structural protein 5A (NS5A) of hepatitis C virus (HCV) has been implicated in inhibition of antiviral activity of IFN. While previous studies have suggested an interaction between NS5A and the double-stranded RNA-dependent protein kinase (PKR), the possibility still remains that interaction with another molecule(s) is involved in the NS5A-mediated inhibition of IFN. In the present study, we investigated a possible interaction between NS5A and 2',5'-oligoadenylate synthetase (2-5AS), another key molecule in antiviral activity. We observed that NS5A physically interacted with 2-5AS in cultured cells, with an N-terminal portion of NS5A [aa 1-148; NS5A(1-148)] and two separate portions of 2-5AS (aa 52-104 and 184-275) being involved in the interaction. Single point mutations at residue 37 of NS5A affected the degree of the interaction with 2-5AS, with a Phe-to-Leu mutation (F37L) augmenting and a Phe-to-Asn mutation (F37N) diminishing it. Virus rescue assay revealed that the full-length NS5A (NS5A-F) and NS5A(1-148), the latter of which contains neither the IFN sensitivity-determining region (ISDR) nor the PKR-binding domain, significantly counteracted the antiviral activity of IFN. Introduction of a F37N mutation into NS5A(1-148) impaired the otherwise more significant IFN-inhibitory activity of NS5A(1-148). It was also found that the F37N mutation was highly disadvantageous for the replication of an HCV RNA replicon. Taken together, our results suggest the possibility that NS5A interacts with 2-5AS and inhibits the antiviral activity of IFN in an ISDR-independent manner.