PA subunit of RNA polymerase as a promising target for anti-influenza virus agents.

RNA polymerase of influenza virus is a specific enzyme necessary for the viral replication. A siRNA against the RNA polymerase and the RNA polymerase inhibitor L-742,001 reduced accumulation of viral RNAs in the infected cells. L-742,001 strongly inhibited virus re-growth after removal of the agent from the culture, whereas the neuraminidase inhibitor zanamivir did not. L-742,001-resistant mutants showed a Thr-20 to Ala substitution in the PA subunit of RNA polymerase. The drug-resistant virus showed a slight reduction in the susceptibility to L-742,001 in both the plaque assay (threefold reduction) and enzyme assay (two- to three-fold reduction). The resistance levels were lower than those of zanamivir-resistant mutants in the plaque assay. Against zanamivir-resistant mutants, L-742,001 retained the same antiviral activity as against the wild-type strain. These results indicate that L-742,001 is most likely to act at the PA subunit, and possesses a unique profile. It is suggested that PA subunit of RNA polymerase is a promising target for anti-influenza virus agents.

Efficacy of quinolones against secondary pneumococcal pneumonia after influenza virus infection in mice.

We established a mouse model of secondary pneumococcal pneumonia after influenza virus infection and investigated the efficacy of several quinolones against pneumonia in this model. Gatifloxacin exhibited the highest efficacy among the quinolones examined and is probably useful for the treatment of secondary bacterial pneumonia.

Characterization of protective immune responses induced by nasal influenza vaccine containing mutant cholera toxin as a safe adjuvant (CT112K).

Immune responses induced by a nasal influenza vaccine with a mutant cholera toxin (CT112K), known to be a safe adjuvant, were characterized in BALB/c mice to confirm the most suitable regimen of this vaccine for humans. Mice received a primary intranasal administration of the adjuvant (0.1 micro g)-combined PR8 vaccine (0.1 micro g) and a secondary administration of the PR8 vaccine alone (0.1 micro g) 4 weeks later. Two weeks after the secondary immunization, the mice were infected with a nonlethal or a lethal dose of PR8 viruses. Nasal and lung wash virus titers 1 or 3 days after infection indicated that complete protection could be provided by secondary immune responses, which had an immediate effect of preventing infection 2 weeks after the secondary immunization. In this two-dose regimen, high levels of secondary IgA, IgG and IgM antibody-forming cell (AFC) responses were induced in the nasal-associated lymphoid tissue and the spleen. In parallel with the AFC responses, high levels of nasal wash anti-PR8 HA IgA, and lung and serum IgG antibody (Ab) responses were induced 2 weeks after the secondary immunization. The two-dose regimen also induced accelerated delayed-type hypersensitivity responses, which exhibited almost the same peak height as that in the case of the primary response. In addition, the two-dose regimen induced a low memory cell activity of cytotoxic T lymphocytes, detected by in vitro culture of spleen cells. Thus, the immediate effect of preventing infection was mainly provided by the secondary Ab responses. Moreover, the levels of nasal wash IgA Abs correlated well with cross-protection against infection with variant viruses in the upper respiratory tract (RT). These results suggest that the major protective factors among Ab and T cell-mediated immune responses, which are induced by the two-dose regimen using CT112K-combined vaccines, are the cross-reactive IgA Abs in the upper RT and the less cross-reactive IgG Abs in the lower RT, and that the two-dose regimen is a suitable vaccination condition for humans.

Persistent infection of influenza virus in irradiated mice and its prevention by intranasal vaccination.

Effects of intranasal administration of influenza vaccine on persistent viral infection in gamma-ray irradiated mice were examined. BALB/c mice were exposed to a sub-lethal dose of gamma-ray (7Gy) and infected intranasally with non-lethal A/PR/8/34 (PR8, H1N1) viruses. The mice irradiated on days 0 or +2 of infection showed a significant weight loss with a slight decrease in survival rate 3 weeks after infection. These mice kept infective viruses in the nasal and/or lung sites even 3 weeks after infection (persistent viral infection) without IgA or IgG antibodies (Abs), although non-irradiated infected mice cleared the virus completely within 2 weeks. On the other hand, the pretreatment with the nasal adjuvant-combined vaccine (3 days before irradiation) prevented the persistent viral infection with the Abs. These results demonstrate that the exacerbation of influenza is induced by the irradiation during early days of infection (0-2 days) and that the exacerbation is prevented by the intranasal vaccination at least 3 days before irradiation.

Protection and antibody responses in different strains of mouse immunized with plasmid DNAs encoding influenza virus haemagglutinin, neuraminidase and nucleoprotein.

Protection against influenza virus infection and antibody responses in mice vaccinated with plasmid DNAs encoding haemagglutinin (HA), neuraminidase (NA) and nucleoprotein (NP) were compared among BALB/c (H-2d), B10 (H-2b) and C3H (H-2k) mice. Mice were inoculated with each DNA construct twice, 3 weeks apart, at a dose of 1 microg per mouse by particle-mediated DNA transfer (gene gun) to the epidermis. They were challenged with a lethal dose of the homologous virus 7 days after the second vaccination. NA-DNA provided significant protection in all strains of mouse, whereas HA-DNA afforded significant protection only in BALB/c mice. The serum antibody titres against NA or HA molecules in BALB/c, C3H and B10 mice were high, intermediate and low, respectively. NP-DNA failed to provide protection in any strain of mouse, and elicited low titres of anti-NP antibodies. These results suggest that NA-DNA can be used as a vaccine component to provide effective protection against influenza virus infection in various strains of mouse.