Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin

Background

Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis.

Methodology/Principal Findings

The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus.

Conclusions/Significance

Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine.     

Anti-androgens with full antagonistic activity toward human prostate tumor LNCaP cells with mutated androgen receptor

Anti-androgens were designed based on the principle of inhibiting the folding of helix 12 of the nuclear androgen receptor. The prepared anti-androgens exhibited full antagonistic activity toward human prostate tumor LNCaP cells with T877A point-mutated nuclear androgen receptor, as far as examined, towards which other known anti-androgens, including hydroxyflutamide, are inactive or act as androgen agonists.     

Novel non-steroidal/non-anilide type androgen antagonists with an isoxazolone moiety

3-Substituted (Z)-4-(4-N,N-dialkylaminophenylmethylene)-5(4H)-isoxazolones and related compounds were designed and prepared as candidates for structurally novel androgen antagonists. Several compounds showed potent anti-androgenic activity as assessed by nuclear androgen receptor binding assay and growth inhibition assay using androgen-dependent Shionogi carcinoma cells SC-3. They were approximately 10--220 times more potent than flutamide in these assay systems. They also showed anti-androgenic activity toward prostate tumor cell line LNCaP, which has an aberrant nuclear androgen receptor.     

Ground based verification of JAXA hardware for life science experiments.

Japanese new space agency (merger of NASDA, ISAS and NAL), JAXA (Japan Aerospace Exploration Agency) has been developing the Plant Experiment Unit (PEU) and the Cell Experiment Unit (CEU), which will be used within the Cell Biology Experiment Facility (CBEF) in "Kibo" Japanese Experiment Module (JEM) of ISS. They can also be operated within the Clean Bench (CB) in Kibo. We report the preliminary results of ground based verification experiments using the PEU and the CEU. Six units of PEU/CEU in microgravity section and 4 units of PEU/CEU in control-g section, will be operated within CBEF.     

Isolation of temperature-sensitive p53 mutations from a comprehensive missense mutation library

Temperature-sensitive (ts) mutations have been used as a genetic and molecular tool to study the functions of many gene products. Each ts mutant protein may contain a temperature-dependent intramolecular mechanism such as ts conformational change. To identify key ts structural elements controlling the protein function, we screened ts p53 mutants from a comprehensive mutation library consisting of 2,314 p53 missense mutations for their sequence-specific transactivity through p53-binding sequences in Saccharomyces cerevisiae. We isolated 142 ts p53 mutants, including 131 unreported ts mutants. These mutants clustered in beta-strands in the DNA-binding domain, particularly in one of the two beta-sheets of the protein, and 15 residues (Thr155, Arg158, Met160, Ala161, Val172, His214, Ser215, Pro223, Thr231, Thr253, Ile254, Thr256, Ser269, Glu271, and Glu285) were ts hot spots. Among the 142 mutants, 54 were examined further in human osteosarcoma Saos-2 cells, and it was confirmed that 89% of the mutants were also ts in mammalian cells. The ts mutants represented distinct ts transactivities for the p53 binding sequences and a distinct epitope expression pattern for conformation-specific anti-p53 antibodies. These results indicated that the intramolecular beta-sheet in the core DNA-binding domain of p53 was a key structural element controlling the protein function and provided a clue for finding a molecular mechanism that enables the rescue of the mutant p53 function.     

Development and verification of hardware for life science experiments in the Japanese Experiment Module "Kibo" on the International Space Station

Japan Aerospace Exploration Agency (JAXA) has developed a cell biology experiment facility (CBEF) and a clean bench (CB) as a common hardware in which life science experiments in the Japanese Experiment Module (JEM known as "Kibo") of the International Space Station (ISS) can be performed. The CBEF, a CO2 incubator with a turntable that provides variable gravity levels, is the basic hardware required to carry out the biological experiments using microorganisms, cells, tissues, small animals, plants, etc. The CB provides a closed aseptic operation area for life science and biotechnology experiments in Kibo. A phase contrast and fluorescence microscope is installed inside CB. The biological experiment units (BEU) are designed to run individual experiments using the CBEF and the CB. A plant experiment unit (PEU) and two cell experiment units (CEU type1 and type2) for the BEU have been developed.     

Quick and reversible inhibition of soybean root nodule growth by nitrate involves a decrease in sucrose supply to nodules

The upper part of a nodulated soybean root hydroponically cultured in a glass bottle was monitored using a computer microscope under controlled environmental conditions, and the diameter of individual nodules was measured from 10-24 d after planting. The diameter of a root nodule attached to the primary root increased from 1 mm to 6 mm for 2 weeks under nitrogen-free conditions. The increase in diameter of the nodules was almost completely stopped after 1 d of supplying 5 mM nitrate, and was due to the cessation of nodule cell expansion. However, nodule growth quickly returned to the normal growth rate following withdrawal of nitrate from the solution. The reversible depression of nodule growth by nitrate was similar to the restriction of photoassimilate supply by continuous dark treatment for 2 d followed by normal light/dark conditions. In addition, the inhibitory effect of nitrate was partially alleviated by the addition of 3% (w/v) sucrose to the medium. Plant leaves were exposed to (11)C or (14)C-labelled carbon dioxide to investigate the effects of 5 mM nitrate on the translocation and distribution of photosynthates to nodules and roots. Supplying 5 mM nitrate stimulated the translocation rate and the distribution of labelled C in nitrate-fed parts of the roots. However, the (14)C partitioning to nodules decreased from 9% to 4% of total (14)C under conditions of 5 mM nitrate supply. These results indicate that the decrease in photoassimilate supply to nodules may be involved in the quick and reversible nitrate inhibition of soybean nodule growth.     

Effects of periodic administration ofNocardia rubra cell-wall skeleton on immunoglobulin production and B-cell-stimulatory factor activity in vitro in workers at a poison gas factory

Summary The former workers at the Okunojima poison gas factory (poison gas workers) are a high-risk group for malignant neoplasms and show abnormalities in cellular immunity. At the same time, poison gas workers often have chronic respiratory diseases, such as chronic bronchitis, and are highly susceptible to respiratory infections. To explore the possibility of immunological cancer prevention, we have periodically administered 200 µgNocardia rubra cell-wall skeleton (N-CWS) to poison gas workers once every 3 months since December 1978. During this period, we noted a significantly lower incidence of influenza among poison gas workers receiving N-CWS than in those not receiving the drug during the influenza epidemic. This finding suggested that the administration of N-CWS enhanced the resistance of these workers to infections. Therefore, periodical administration of N-CWS to poison gas workers was considered to enhance the reduced T-cell function of normalizing antibody production by stimulating the production of B-cell-stimulatory factor (BSF). In the present study, to clarify the mechanism of immunosuppression in the poison gas workers and to examine the effects of continual administration of N-CWS on this condition, we compared the immunoglobulin production and the proliferative and differentiative activities of B-cell-stimulatory factor (BSF) of peripheral blood mononuclear cells (PBMC), in poison gas workers treated or not treated with N-CWS. Comparisons were also made with age-matched healthy controls. In the untreated poison gas workers, immunoglobulin and BSF production of PBMC were reduced as compared with the control group. On the other hand, in the poison gas workers receiving N-CWS, immunoglobulin and BSF production of PBMC were restored nearly to the control level. These results show that in vitro antibody production in the poison gas workers was reduced and that a reduction in BSF production of T cells was one of its causes.     

Effects of soil structural discontinuity on root and shoot growth and water use of maize

The role of roots penetrating various undisturbed soil horizons beneath loose layer in water use and shoot growth of maize was evaluated in greenhouse experiment. 18 undisturbed soil columns 20 cm in diameter and 20 cm in height were taken from the depths 30–50 cm and 50–70 cm from a Brown Lowland soil, a Pseudogley and a Brown Andosol (3 columns from each depth and soil). Initial resistance to penetration in undisturbed soil horizons varied from 2.5 to 8.9 MPa while that in the loose layer was 0.01 MPa. The undisturbed horizons had a major effect on vertical arrangement of roots. Root length density in loose layer varied from 96 to 126 km m^-3 while in adjacent stronger top layers of undisturbed horizons from 1.6 to 20.0 km m^-3 with higher values in upper horizons of each soil. For specific root length, the corresponding ranges were 79.4–107.7 m g^-1 (on dry basis) and 38.2–63.7 m g^-1, respectively. Ratios of root dry weight per unit volume of soil between loose and adjacent undisturbed layers were much lower than those of root length density indicating that roots in undisturbed horizons were produced with considerably higher partition of assimilates. Root size in undisturbed horizons relative to total roots was from 1.1 to 38.1% while water use from the horizons was from 54.1 to 74.0%. Total water use and shoot growth were positively correlated with root length in undisturbed soil horizons. There was no correlation between shoot growth and water use from the loose layers.     

Significance of Spermidine in the Initiation of Adventitious Buds in Stem Segments of Torenia

When stem segments of Torenia fournieri Lind. were culturedin vitro, the initiation of adventitious buds, which is usuallyinduced by cytokinin, was induced by application of polyamines,such as putrescine and spermidine. Addition of arginine hada slight inductive effect. When cyclohexylamine, an inhibitorof spermidine synthase, was added simultaneously with putrescine,induction of the initiation of buds by putrescine was stronglyinhibited. However, application of the inhibitor together withspermidine had no effect on the spermidine-induced initiationof buds. The induction of initiation of buds by a cytokinin,by a calcium ionophore, by cyclic AMP, and by a phorbol ester,which was accompanied in each case by elevation of the levelsof endogenous spermidine, was also inhibited by cyclohexylamine.These results suggest the involvement of spermidine in the initiationof adventitious buds in stem segments of Torenia. ²Present address: Radiation Effects Research Foundation, Hijiyamakouen5-2, Minami-ku, Hiroshima, 732 Japan.