Joe Chiba

Synthetic double-stranded RNA poly(I:C) combined with mucosal vaccine protects against influenza virus infection.

ABSTRACT The mucosal adjuvant effect of synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I:C)] against influenza virus was examined under intranasal coadministration with inactivated hemagglutinin (HA) vaccine in BALB/c mice and was shown to have a protective effect against both nasal-restricted infection and lethal lung infection. Intranasal administration of vaccine from PR8 (H1N1) with poly(I:C) induced a high anti-HA immunoglobulin A (IgA) response in the nasal wash and IgG antibody response in the serum, while vaccination without poly(I:C) induced little response. Intracerebral injection confirmed the safety of poly(I:C). In addition, we demonstrated that administration of poly(I:C) with either A/Beijing (H1N1) or A/Yamagata (H1N1) vaccine conferred complete protection against PR8 challenge in this mouse nasal infection model, suggesting that poly(I:C) possessed cross-protection ability against variant viruses. To investigate the mechanism of the protective effect of poly(I:C), mRNA levels of Toll-like receptors and cytokines were examined in the nasal-associated lymphoid tissue after vaccination or virus challenge. Intranasal administration of HA vaccine with poly(I:C) up-regulated expression of Toll-like receptor 3 and alpha/beta interferons as well as Th1- and Th2-related cytokines. We propose that poly(I:C) is a new effective intranasal adjuvant for influenza virus vaccine.

Nuclear localization of the truncated hepatitis C virus core protein with its hydrophobic C terminus deleted.

The core protein of hepatitis C virus (HCV) is considered to be cleaved from the N terminus of the large precursor polyprotein by cellular signalase. The HCV cDNA encoding the core protein was expressed (i) in monkey COS cells by a plasmid expression vector driven by the SR alpha promoter, and (ii) in insect cells by a recombinant baculovirus. The expressed product had an M(r) of 22,000 and was located in the cytoplasm. When the C-terminal hydrophobic domains were deleted, however, the truncated core proteins were translocated into the nucleus. The truncated core proteins were located in the nucleus even when they were expressed as a fusion protein with E. coli beta-galactosidase, which is essentially localized in the cytoplasm. Plasmids containing HCV cDNAs with a deletion in one of the regions encoding clusters of basic amino acids were expressed in COS cells and the localization of the core protein was examined. The residues PRRGPR were suggested to play an important role in nuclear localization. HCV is an RNA virus and its life cycle was originally considered to be confined to the cytoplasm; the present study, however, suggests that the HCV core protein can translocate into the nucleus under certain circumstances.

Cross-Protection against H5N1 Influenza Virus Infection Is Afforded by Intranasal Inoculation with Seasonal Trivalent Inactivated Influenza Vaccine

Abstract Background. Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice. Methods. BALB/c mice were inoculated 3 times, either intranasally or subcutaneously, with the trivalent inactivated influenza vaccine licensed in Japan for the 2005–2006 season. The vaccine included A/NewCaledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2), and B/Shanghai/361/2002 viral strains and was administered together with poly(I):poly(C12U) (Ampligen) as an adjuvant. At 14 days after the final inoculation, the inoculated mice were challenged with either the A/HongKong/483/97, the A/Vietnam/1194/04, or the A/Indonesia/6/05 strain of H5N1 influenza virus. Results. Compared with noninoculated mice, those inoculated intranasally manifested cross-reactivity of mucosal IgA and serum IgG with H5N1 virus, as well as both a reduced H5N1 virus titer in nasal-wash samples and increased survival, after challenge with H5N1 virus. Subcutaneous inoculation did not induce a cross-reactive IgA response and did not afford protection against H5N1 viral infection. Conclusions. Intranasal inoculation with annual influenza vaccine plus the Toll-like receptor—3 agonist, poly(I): poly(C12U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.

Improvement in the basic technology of electrofusion for generation of antibody-producing hybridomas

In order to define the optimum conditions of electrofusion technique for the generation of antibody-producing hybridomas, mouse spleen cells or EBV-transformed human B cells were fused with mouse myeloma cells (SP2/0) or human fusion partner cells (KR-4 or KR-12), respectively, by electric field pulse under various conditions. The results confirm reports that the presence of both Ca2+ and Mg2+ in fusion medium and pretreatment of mixed cells with proteases improve hybridoma yield. Moreover, the presence of liposome or hydrophobic protein in the fusion medium greatly enhanced the yield. Under optimum conditions, hybridoma yields of mouse cells and human cells were 2.5 X 10(-4) and 1 X 10(-4), respectively. These efficiencies were about ten times higher than those obtained by the conventional polyethylene glycol technique. Microscopic observation of the fusion-process revealed that in a human cell system 20%-50% of the cells were physically fused, although only one in 5000 physically fused human cells grew as a hybridoma after hypoxanthine-aminopterin-thymidine selection.

Protection against influenza virus infection by intranasal administration of C3d-fused hemagglutinin.

For the induction of mucosal immune responses by intranasal vaccination, cholera toxin B subunits (CTB) and Escherichia coli heat-labile toxin (LT) are often administered as mucosal adjuvants in order to enhance immune responses to mucosally co-administered bystander antigens. However, these toxin also are the causative agents of diarrhea. There is a demand for the establishment of an effective and safer adjuvant or vaccine that elicits mucosal immunity, but does not require the use of CTB or LT adjuvants. In order to induce protective mucosal immune responses in the nasal area against influenza virus infection, we have examined the recombinant protein composed of the complement component, C3d, which is fused to the secreted form of hemagglutinin (sHA-mC3d3) in the influenza-BALB/c mouse model. The fusion protein sHA-mC3d3, the secretory form of hemagglutinin, and the transmembrane form of HA (tmHA) from the influenza virus were intranasally administered to the mice with or without CTB containing a trace amount of holotoxin (CTB*) as an adjuvant. After intranasal administration of these proteins with CTB*, all mice produced nasal IgA and serum IgG antibodies (Abs) against the viral HA. In addition, viral infection was completely inhibited in these mice. In contrast, in the absence of the adjuvant, only sHA-mC3d3-induced locally secreted IgA and serum IgG Abs and provided complete protection against the influenza virus challenge. Thus, C3d fused to the influenza HA antigen is an effective and safe tool for mucosal vaccination.

A sensitive enzyme-linked immunosorbent assay of ochratoxin A based on monoclonal antibodies.

We prepared seven monoclonal antibodies (mAbs, OTA.1, 2, 3, 4, 5, 6 and 7) which were reacted with ochratoxin A (OTA), and have developed a specific and highly sensitive enzyme-linked immunosorbent assay (ELISA) for detection of OTA. The mAbs, OTA.1, 3, 4, 5 and 7, specifically reacted with OTA but much less with its analogs, ochratoxin B (OTB, about 1% of OTA) and ochratoxin alpha (OT alpha, less than 0.1% of OTA). One of the mAbs, OTA.2, equally reacted with OTA and OTB but hardly at all with (4R)-4-hydroxyochratoxin A or OT alpha (less than 0.1% of OTA). All of the mAbs reacted with ochratoxin C. None of the mAbs reacted with coumarin, 4-hydroxycoumarin or L-beta-phenylalanine. In the competitive ELISA with OTA.1 and OTA.7, the lowest detectable amount of standard OTA in solution was 50 pg/ml (2.5 pg per assay). This assay was applied for the quantitation of OTA added to chicken meat, wheat flour, porcine plasma and bovine serum. With minimal sample preparation, reliable and reproducible determinations were possible when concentrations of OTA were higher than 0.1-1 ng/g.

A new strategy of gene trapping in ES cells using 3'RACE

Abstract“Gene trapping” in embryonic stem (ES) cells is a novel approach to identify a series of genes in mammals concomitant with the production of the corresponding mutant mice. However, this approach is currently unable to identify genes that are not expressed in ES cells. Here we describe a strategy to identify gene trapping clones which is not based on expression of a reporter gene. It uses theneor gene which lacks a polyadenylation signal and has a splice donor signal. Expression of theneor gene as fusion transcripts with the 3′ end containing the polyadenylation signal of tagged genes allows the identification of these clones by 3′ rapid amplification of the cDNA end in undifferentiated ES cells, even if the genes are not expressed in ES cells. Amplification was observed in about 25% of G418-resistant clones. Sequence analyses suggested the amplifications represent gene trapping events. The feasibility of this approach was further assessed by analysing one clone, PAT-12, in detail.

Intranasal administration of adjuvant-combined vaccine protects monkeys from challenge with the highly pathogenic influenza A H5N1 virus.

The effectiveness in cynomolgus macaques of intranasal administration of an influenza A H5N1 pre‐pandemic vaccine combined with synthetic double‐stranded RNA (polyI/polyC12U) as an adjuvant was examined. The monkeys were immunized with the adjuvant‐combined vaccine on weeks 0, 3, and 5, and challenged with the homologous virus 2 weeks after the third immunization. After the second immunization, the immunization induced vaccine‐specific salivary IgA and serum IgG antibodies, as detected by ELISA. The serum IgG antibodies present 2 weeks after the third immunization not only had high neutralizing activity against the homologous virus, they also neutralized significantly heterologous influenza A H5N1 viruses. The vaccinated animals were protected completely from the challenge infection with the homologous virus. These results suggest that intranasal immunization with the Double stranded RNA‐combined influenza A H5N1 vaccine induce mucosal IgA and serum IgG antibodies which could protect humans from homologous influenza A H5N1 viruses which have a pandemic potential. J. Med. Virol. 82:1754–1761, 2010.

Tetrahymena 14-nm filament-forming protein has citrate synthase activity.

The Tetrahymena 14-nm filament-forming protein (49K protein) is a structural protein involved in oral morphogenesis and in pronuclear behavior during conjugation. Cloning the 49K protein gene from a Tetrahymena thermophila cDNA library, we found that its primary structure exhibits a high sequence identity (51.5%) with porcine heart citrate synthase and retains functional domains. The 49K protein actually possesses citrate synthase activity, and is detected in mitochondria. These results suggest that the 49K protein has dual functions as both a respiratory enzyme and a structural protein in the cytoskeleton.

Nuclear Pore Complex Protein Mediated Nuclear Localization of Dicer Protein in Human Cells

Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.