A recombinant baculovirus expressing the hemagglutinin gene of the influenza virus, A/PR/8/34 (H1N1), under the control of the chicken β-actin promoter, was constructed. To determine the induction of protective immunity in vivo, mice were inoculated with the recombinant baculovirus by intramuscular, intradermal, i.p., and intranasal routes and then were challenged with a lethal dose of the influenza virus. Intramuscular or i.p. immunization with the recombinant baculovirus elicited higher titers of antihemagglutinin Ab than intradermal or intranasal administration. However, protection from a lethal challenge of the influenza virus was only achieved by intranasal immunization of the recombinant baculovirus. Surprisingly, sufficient protection from the lethal influenza challenge was also observed in mice inoculated intranasally with a wild-type baculovirus, as evaluated by reductions in the virus titer, inflammatory cytokine production, and pulmonary consolidations. These results indicate that intranasal inoculation with a wild-type baculovirus induces a strong innate immune response, which protects mice from a lethal challenge of influenza virus.

No abstract

Wild-type Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a variety of mammalian cell types in vitro, but does not replicate in these cells. We investigated the effects of AcMNPV in the induction of the immune response and tumor metastasis in mice. After intravenous injection, AcMNPV was taken up by the liver and spleen, and preferentially infected dendritic cells (DCs) and B cells in the spleen; costimulatory molecules CD40, CD80, and CD86 were upregulated in the DCs. The hepatic mononuclear cells (MNCs) in these animals were highly cytotoxic to natural killer (NK)-sensitive YAC-1 and B16 melanoma cells, but not to NK-resistant EL4 cells. Intravenous injection of AcMNPV-induced NK cell proliferation in the liver and spleen, and enhanced antitumor immunity in mice with B16 liver metastases. Furthermore, such treatment increased the survival of C57BL/6, J alpha 281 (-/-), and interferon (IFN)-gamma (-/-) mice that were previously injected with B16 tumor cells. AcMNPV injection did not enhance the survival of NK cell-depleted mice. Moreover, one AcMNPV treatment effectively prolonged survival in a B16 liver metastasis model, and was equivalent to five treatments with recombinant interleukin-12 (IL-12) protein. These findings suggest that AcMNPV efficiently stimulates NK cell-mediated antitumor immunity.

We have investigated the ability of antisense phosphorothioate oligonucleotides to enhance the survival of mice infected with influenza A virus. The oligonucleotides were complementary to sequences surrounding the translation initiation codons of the viral PB2 or PA genes (PB2-as or PA-as, respectively) of the influenza A virus RNA polymerases. Intravenous administration of PB2-as in a complex with a cationic liposome, Tfx-10, significantly prolonged the mean survival time in days and increased overall survival rates of mice infected with the influenza A virus. Liposomally encapsulated PB2-as inhibited viral growth in lung tissues and reduced pulmonary consolidations. Liposomally encapsulated PB2-as could be an effective therapeutic agent against influenza A virus.

No abstract

The inhibition of specific transcription regulatory proteins is a new approach to control gene expression. The transcriptional activities of DNA-binding proteins can be inhibited by the use of double-stranded oligonucleotides that compete for the binding to their specific target sequences in promoters and enhancers. We used nicked (NDODN-kappaB) and circular (CDODN-kappaB) dumbbell DNA oligonucleotides containing a NF-kappaB binding site to analyze the inhibition of the NF-kappaB-dependent activation of the human immunodeficiency virus type-1 (HIV-1) enhancer. The dumbbell DNA oligonucleotides are stable, short segments of double-stranded DNA with closed nucleotide loops on each end, which confer resistance to exonucleases. The dumbbell and other oligonucleotides (decoys) with the NF-kappaB sequence were found to compete with the native strand for NF-kappaB binding. The circular dumbbell and double-stranded phosphorothioate oligonucleotides competed with the native strand for binding to the NF-kappaB binding proteins, while the nicked NF-kappaB dumbbell was a less effective competitor. In Jurkat T-cells, the dumbbell and other oligonucleotides were tested for their ability to block the activation of the plasmid HIV-NL4-3 Luc. The CDODN-kappaB strongly inhibits the specific transcriptional regulatory proteins, as compared with the NDODN-kappaB and the double stranded phosphodiester oligonucleotides. On the other hand, the double stranded phosphorothioate oligonucleotides could also block this activation, but the effect was non-specific. The circular (CDODN) dumbbell oligonucleotides may efficiently compete for the binding of specific transcription factors within cells, thus providing anti-HIV-1 or other therapeutic effects.

DNA enzymes are RNA-cleaving single-stranded DNA molecules. We designed DNA enzymes targeting the PB2 mRNA translation initiation (AUG) region of the in£uenza A virus (A/PR/8/34). The modi¢ed DNA enzymes have one or two N3P P-P5P P phosphoramidate bonds at both the 3P P-and 5P Ptermini of the oligonucleotides, which signi¢cantly enhanced their nuclease resistance. These modi¢ed DNA enzymes had the same cleavage activity as the unmodi¢ed DNA enzymes, determined by kinetic analyses, and reduced in£uenza A virus replication by more than 99%, determined by plaque formation. These DNA enzymes are highly speci¢c; their protective e¡ect was not observed in in£uenza B virus (B/Ibaraki)-infected Madin^Darby canine kidney cells.

In order to define neutralization regions on the envelope antigen of human T-cell leukemia virus type I (HTLV-I), we have generated a number of new anti-envelope gp46 monoclonal antibodies from rats and mice. Epitopes recognized by new monoclonal antibodies which could neutralize HTLV-I in syncytium and transformation inhibition assays were localized to sequences in gp46 from amino acids 186 to 193, 190 to 195, 191 to 195, 191 to 196, and 194 to 199. Ovalbumin-conjugated synthetic gp46 peptides containing these neutralization epitopes, pepl90-199 (a synthetic gp46 peptide containing amino acids 190 to 199) and pepl80-204, but not pepl85-194 or pepl94-203, could give rise to HTLV-I-neutralizing antibody responses in rabbits. These immune or nonimmune rabbits were then challenged with HTLV-I by intravenous inoculation with 5 x 107 live HTLV-I-producing ILT-8M2 cells. By a PCR assay, it was revealed that HTLV-I provirus was detected in peripheral blood lymphocytes from nonimmune and pep288-312-immunized rabbits, whereas the provirus was not detected in peripheral blood lymphocytes from pepl90-199and pepl80-204-immunized rabbits over an extended period. These results suggest that the induction of anti-gp46 neutralizing antibody responses by immunization with synthetic peptides has the potential to protect animals against HTLV-I infection in vivo.