A bio-nanocapsule containing envelope protein domain III of Japanese encephalitis virus protects mice against lethal Japanese encephalitis virus infection.

An engineered bio-nanocapsule (BNC) comprising modified hepatitis B surface antigen L protein was used as a physical scaffold for envelope protein domain III (D3) of Japanese encephalitis virus (JEV). At the N terminus, the BNC contained a two-tandem repeat of the Z domain (ZZ) derived from Staphylococcus aureus protein A (ZZ-BNC). The Lys-rich ZZ moiety exposed on the surface of ZZ-BNC was used for chemical conjugation with the JEV D3 antigen, which had been expressed and purified from Escherichia coli. Immunization of mice with D3 loaded on the surface of ZZ-BNC (ZZ-BNC:D3) augmented serum IgG response against JEV and increased protection against lethal JEV infection. The present study suggests that innocuous recombinant antigens, when loaded on the surface of ZZ-BNC, can be transformed to immunogenic antigens.

Anti-adult T-cell leukemia effects of brown algae fucoxanthin and its deacetylated product, fucoxanthinol.

Adult T-cell leukemia (ATL) is a fatal malignancy of T lymphocytes caused by human T-cell leukemia virus type 1 (HTLV-1) infection and remains incurable. Carotenoids are a family of natural pigments and have several biological functions. Among carotenoids, fucoxanthin is known to have antitumorigenic activity, but the precise mechanism of action is not elucidated. We evaluated the anti-ATL effects of fucoxanthin and its metabolite, fucoxanthinol. Both carotenoids inhibited cell viability of HTLV-1-infected T-cell lines and ATL cells, and fucoxanthinol was approximately twice more potent than fucoxanthin. In contrast, other carotenoids, beta-carotene and astaxanthin, had mild inhibitory effects on HTLV-1-infected T-cell lines. Importantly, uninfected cell lines and normal peripheral blood mononuclear cells were resistant to fucoxanthin and fucoxanthinol. Both carotenoids induced cell cycle arrest during G(1) phase by reducing the expression of cyclin D1, cyclin D2, CDK4 and CDK6, and inducing the expression of GADD45alpha, and induced apoptosis by reducing the expression of Bcl-2, XIAP, cIAP2 and survivin. The induced apoptosis was associated with activation of caspase-3, -8 and -9. Fucoxanthin and fucoxanthinol also suppressed IkappaBalpha phosphorylation and JunD expression, resulting in inactivation of nuclear factor-kappaB and activator protein-1. Mice with severe combined immunodeficiency harboring tumors induced by inoculation of HTLV-1-infected T cells responded to treatment with fucoxanthinol with suppression of tumor growth, showed extensive tissue distribution of fucoxanthinol, and the presence of therapeutically effective serum concentrations of fucoxanthinol. Our preclinical data suggest that fucoxanthin and fucoxanthinol could be potentially useful therapeutic agents for patients with ATL.

Tricomponent fusion complex comprising a viral antigen, a pentameric α-helical coiled-coil, and an immunoglobulin-binding domain as an effective antiviral vaccine.

The pentameric coiled-coil domain of cartilage oligomeric matrix protein (COMP) genetically fused to the Z domain of Staphylococcus aureus protein A, an immunoglobulin-binding domain (IBD), was evaluated as a viral antigen carrier complex. In a proof-of-concept study, recombinant Japanese encephalitis virus (JEV) E protein domain III (D3) was loaded onto the COMP-Z fusion protein by chemical conjugation, and the tricomponent complex generated, COMP-Z/D3, was evaluated for its vaccine efficacy in a mouse JEV infection model. Immunization with the complex conferred substantially greater protection against lethal JEV infection than the unloaded antigen. Next, a tricomponent complex was engineered in which the three molecular entities (the D3 antigen, COMP coiled-coil domain, and Z domain) were genetically connected in tandem to create the D3-COMP-Z tricomponent complex, or its reversal oriented construct, Z-COMP-D3. The fusion complexes were produced as inclusion bodies in Escherichia coli, but could be refolded to biologically active pentamers that retained the E protein antigenicity and the IBD function. Immunization with the refolded complexes conferred a high level of protection against lethal JEV infection, similar in efficacy to that of the tricomponent complex generated by chemical conjugation. These results demonstrate that the tricomponent complex, whether generated by chemical or genetic fusion, is a promising molecular design for the creation of effective subunit vaccines against viral infections.

Oral Immunization Against Candidiasis Using Lactobacillus casei Displaying Enolase 1 from Candida albicans.

Candidiasis is a common fungal infection that is prevalent in immunocompromised individuals. In this study, an oral vaccine against Candida albicans was developed by using the molecular display approach. Enolase 1 protein (Eno1p) of C. albicans was expressed on the Lactobacillus casei cell surface by using poly-gamma-glutamic acid synthetase complex A from Bacillus subtilis as an anchoring protein. The Eno1p-displaying L. casei cells were used to immunize mice, which were later challenged with a lethal dose of C. albicans. The data indicated that the vaccine elicited a strong IgG response and increased the survival rate of the vaccinated mice. Furthermore, L. casei acted as a potent adjuvant and induced high antibody titers that were comparable to those induced by strong adjuvants such as the cholera toxin. Overall, the molecular display method can be used to rapidly develop vaccines that can be conveniently administered and require minimal processing.

An oral vaccine against candidiasis generated by a yeast molecular display system.

Enolase 1 (Eno1p) of Candida albicans is an immunodominant antigen. However, conventional technologies for preparing an injectable vaccine require purification of the antigenic protein and preparation of an adjuvant. To develop a novel type of oral vaccine against candidiasis, we generated Saccharomyces cerevisiae cells that display the Eno1p antigen on their surfaces. Oral delivery of the engineered S. cerevisiae cells prolonged survival rate of mice that were subsequently challenged with C. albicans. Given that a vaccine produced using molecular display technology avoids the need for protein purification, this oral vaccine offers a promising alternative to the use of conventional and injectable vaccines for preventing a range of infectious diseases.

Anti-neoplastic effects of fucoxanthin and its deacetylated product, fucoxanthinol, on Burkitt's and Hodgkin's lymphoma cells.

Fucoxanthin (FX) is a natural carotenoid with reported antitumorigenic activity. This study explored the effects of FX and its deacetylated product, fucoxanthinol (FXOH), on B-cell malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma and Epstein-Barr virus-immortalized B cells. Both FX and FXOH reduced the viability of these malignant B cells in a dose-dependent manner accompanied by the induction of cell cycle arrest during G1 phase and caspase-dependent apoptosis. FXOH was approximately twice more potent than FX in these activities. In contrast, normal peripheral blood mononuclear cells were resistant to FX and FXOH. Strong and constitutive activation of nuclear factor-κB (NF-κB) is a common characteristic of many B-cell malignancies, and FXOH suppressed constitutive NF-κB activity. NF-κB inhibition was accompanied by downregulation of NF-κB-dependent anti-apoptotic and cell cycle regulator gene products, including Bcl-2, cIAP-2, XIAP, cyclin D1 and cyclin D2. The results indicated that FX and FXOH are potentially useful therapeutic agents in B-cell malignancies characterized by aberrant regulation of NF-κB.

Japanese encephalitis virus structural and nonstructural proteins expressed in Escherichia coli induce protective immunity in mice.

Ectodomain of Japanese encephalitis virus (JEV) E protein [domains I through III (D1-3), domains I and II (D1-2) and domain III (D3)] and the nonstructural protein 1 (NS1) were expressed in Escherichia coli, and administered to BALB/c mice via the intranasal (i.n.) route. The E protein, but not the NS1, induced JEV-specific serum IgG with virus-neutralization capacity in vitro. When mice were lethally challenged with JEV, i.n. immunization with D1-3, D1-2, D3, or a mouse brain-derived formalin-inactivated JE vaccine conferred complete protection, while an 80% protection rate was observed in the NS1 immunized mice. Cytokine analysis of the cervical lymph nodes of mice i.n. immunized with D1-3 or NS1 revealed antigen-specific IL-2 and IL-17 responses, but no IFN-γ T cell response, were observed. This study demonstrates for the first time the i.n. vaccine efficacy of the E. coli-expressed recombinant JEV proteins.

Potential mechanism of resistance to TRAIL-induced apoptosis in Burkitt's lymphoma.

OBJECTIVES: Members of the tumor necrosis factor family are potent inducers of apoptosis in sensitive cells and may be suitable for novel anti-cancer therapies aimed at inducing apoptosis via the activation of receptors with the death domain on malignant cells. We characterized the sensitivity of Burkitt's lymphoma (BL) cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and anti-Fas agonist, and investigated the mechanism of resistance of BL cell lines to TRAIL and Fas apoptotic pathways. METHODS: Epstein-Barr virus (EBV) status in BL cell lines was determined by PCR. The extent of apoptosis following exposure to TRAIL and anti-Fas agonist was measured by 7A6 antigen staining. Expression of TRAIL receptors and Fas was determined by flow cytometry and reverse transcriptase-PCR. Western blot analyses were used to determine the expression of proapoptotic and antiapoptotic proteins. NF-kappaB activity was evaluated by electrophoretic mobility shift assay. RESULTS: The sensitivity of BL cell lines to anti-Fas agonist depended on the expression of Fas. In contrast, the expression of TRAIL receptors did not correlate with the sensitivity to TRAIL-induced apoptosis. Interestingly, EBV-infected BL cell lines which showed constitutive levels of NF-kappaB activation, were TRAIL-resistant. NF-kappaB inhibitors reversed the resistance to TRAIL-induced apoptosis. CONCLUSIONS: Our results suggest that activation of NF-kappaB by EBV infection plays an important role in resistance of BL cell lines to TRAIL-induced apoptosis, and that NF-kappaB inhibitors may be useful adjuncts in clinical use of TRAIL against BL.