The mortality of human infection by influenza A/H5N1 virus can exceed 80%. The high mortality and its poor response to the neuraminidase inhibitor oseltamivir have been attributed to uncontrolled virus-induced cytokine storm. We challenged BALB/c mice with 1,000 LD 50 of influenza A/Vietnam/1194/04. Survival, body weight, histopathology, inflammatory markers, viral loads, T lymphocyte counts, and neutralizing antibody response were documented in infected mice treated individually or in combination with zanamvir, celecoxib, gemfibrozil, and mesalazine. To imitate the real-life scenario, treatment was initiated at 48 h after viral challenge. There were significant improvements in survival rate (P ‫؍‬ 0.02), survival time (P < 0.02), and inflammatory markers (P < 0.01) in the group treated with a triple combination of zanamivir, celecoxib, and mesalazine when compared with zanamivir alone. Zanamivir with or without immunomodulators reduced viral load to a similar extent. Insignificant prolongation of survival was observed when individual agents were used alone. Significantly higher levels of CD4 ؉ and CD8 ؉ T lymphocytes and less pulmonary inflammation were also found in the group receiving triple therapy. Zanamivir alone reduced viral load but not inflammation and mortality. The survival benefits of adding celecoxib and mesalazine to zanamivir could be caused by their synergistic effects in reducing cytokine dysfunction and preventing apoptosis. Combinations of a neuraminidase inhibitor with these immunomodulators should be considered in randomized controlled treatment trials of patients suffering from H5N1 infection.zanamivir ͉ celecoxib ͉ mesalazine

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease. The potential recurrence of the disease from animal reservoirs highlights the significance of development of safe and efficient vaccines to prevent a future SARS epidemic. In this study, we expressed the recombinant receptor-binding domain (rRBD) in mammalian (293T) cells, insect (Sf9) cells, and E. coli, respectively, and compared their immunogenicity and protection against SARS-CoV infection in an established mouse model. Our results show that all rRBD proteins expressed in the above systems maintained intact conformation, being able to induce highly potent neutralizing antibody responses and complete protective immunity against SARS-CoV challenge in mice, albeit the rRBD expressed in 293T cells elicited stronger humoral immune responses with significantly higher neutralizing activity (P < 0.05) than those expressed in Sf9 and E. coli cells. These results suggest that all three rRBDs are effective in eliciting immune responses and protection against SARS-CoV and any of the above expression systems can be used for production of rRBD-based SARS subunit vaccines. Preference will be given to rRBD expressed in mammalian cells for future evaluation of the vaccine efficacy in a non-human primate model of SARS because of its ability to refold into a native conformation more readily and to induce higher level of neutralizing antibody responses than those expressed in E. coli and insect cells.

We have previously reported that a subunit protein vaccine based on the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein and a recombinant adeno-associated virus (rAAV)-based RBD (RBD-rAAV) vaccine could induce highly potent neutralizing Ab responses in immunized animals. In this study, systemic, mucosal, and cellular immune responses and long-term protective immunity induced by RBD-rAAV were further characterized in a BALB/c mouse model, with comparison of the i.m. and intranasal (i.n.) routes of administration. Our results demonstrated that: 1) the i.n. vaccination induced a systemic humoral immune response of comparable strength and shorter duration than the i.m. vaccination, but the local humoral immune response was much stronger; 2) the i.n. vaccination elicited stronger systemic and local specific cytotoxic T cell responses than the i.m. vaccination, as evidenced by higher prevalence of IL-2 and/or IFN-γ-producing CD3+/CD8+ T cells in both lungs and spleen; 3) the i.n. vaccination induced similar protection as the i.m. vaccination against SARS-CoV challenge in mice; 4) higher titers of mucosal IgA and serum-neutralizing Ab were associated with lower viral load and less pulmonary pathological damage, while no Ab-mediated disease enhancement effect was observed; and 5) the vaccination could provide long-term protection against SARS-CoV infection. Taken together, our findings suggest that RBD-rAAV can be further developed into a vaccine candidate for prevention of SARS and that i.n. vaccination may be the preferred route of administration due to its ability to induce SARS-CoV-specific systemic and mucosal immune responses and its better safety profile.

BackgroundPregnant women infected by the pandemic influenza A (H1N1) 2009 virus had more severe disease and higher mortality but its pathogenesis is still unclear.Principal FindingsWe showed that higher mortality, more severe pneumonitis, higher pulmonary viral load, lower peripheral blood T lymphocytes and antibody responses, higher levels of proinflammatory cytokines and chemokines, and worse fetal development occurred in pregnant mice than non-pregnant controls infected by either wild type (clinical isolate) or mouse-adapted mutant virus with D222G substitution in hemagglutinin. These disease-associated changes and the lower respiratory tract involvement were worse in pregnant mice challenged by mutant virus. Though human placental origin JEG-3 cell line could be infected and proinflammatory cytokines or chemokines were elevated in amniotic fluid of some mice, no placental or fetal involvement by virus were detected by culture, real-time reverse transcription polymerase chain reaction or histopathological changes. Dual immunofluorescent staining of viral nucleoprotein and type II alveolar cell marker SP-C protein suggested that the majority of infected alveolar epithelial cells were type II pneumocytes.ConclusionThe adverse effect of this pandemic virus on maternal and fetal outcome is largely related to the severe pulmonary disease and the indirect effect of inflammatory cytokine spillover into the systemic circulation.

Citrobacter rodentium belongs to the attaching and effacing family of enteric bacterial pathogens that includes both enteropathogenic and enterohemorrhagic Escherichia coli. These bacteria infect their hosts by colonizing the intestinal mucosal surface and intimately attaching to underlying epithelial cells. The abilities of these pathogens to exploit the cytoskeleton and signaling pathways of host cells are well documented, but their interactions with the host's antimicrobial defenses, such as inducible nitric oxide synthase (iNOS), are poorly understood. To address this issue, we infected mice with C. rodentium and found that iNOS mRNA expression in the colon significantly increased during infection. Immunostaining identified epithelial cells as the major source for immunoreactive iNOS. Finding that nitric oxide (NO) donors were bacteriostatic for C. rodentium in vitro, we examined whether iNOS expression contributed to host defense by infecting iNOS-deficient mice. Loss of iNOS expression caused a small but significant delay in bacterial clearance without affecting tissue pathology. Finally, immunofluorescence staining was used to determine if iNOS expression was localized to infected cells by staining for the C. rodentium virulence factor, translocated intimin receptor (Tir), as well as iNOS. Interestingly, while more than 85% of uninfected epithelial cells expressed iNOS, fewer than 15% of infected (Tir-positive) cells expressed detectable iNOS. These results demonstrate that both iNOS and intestinal epithelial cells play an active role in host defense during C. rodentium infection. However, the selective expression of iNOS by uninfected but not infected cells suggests that this pathogen has developed mechanisms to locally limit its exposure to host-derived NO.

In Ontario, 1-year survival rates after an index stroke are higher for those from the richest compared to the least wealthy areas, and this is only partly explained by age, sex, comorbid conditions, and other baseline risk factors.

Development of vaccines against severe acute respiratory syndrome (SARS) coronavirus (SARSCoV) is crucial in the prevention of SARS reemergence. The receptor-binding domain (RBD) of SARS-CoV spike (S) protein is an important target in developing safe and effective SARS vaccines. Our previous study has demonstrated that vaccination with adeno-associated virus encoding RBD (RBD-rAAV) induces high titer of neutralizing antibodies. In this study, we further assessed the immune responses and protective effect of the immunization with RBD-rAAV prime/RBD-specific T cell peptide boost, as compared to those of vaccinations with RBD-rAAV and RBD-peptides alone. Our results indicated that RBD-rAAV prime/RBD-peptide boost induced similar Th1 and neutralizing antibody responses, but stronger Th2 and CTL responses than RBD-rAAV prime/boost. The resulting immune responses protected the vaccinated mice from subsequent SARS-CoV challenge, which was evidenced by lower level of viral replication in mouse lung tissues. However, no significant immune responses and protective effect were detected in mice vaccinated with RBDpeptides or blank AAV alone. Since T cell epitopes are highly conserved and boosting with peptides may induce the production of effector memory T cells, our results suggest that the vaccination protocol used may be ideal for providing effective, universal and long-term protection against SARSCoV infection.

Interleukin-17 (IL-17), a member of the IL-17 cytokine family, plays a crucial role in mediating the immune response against extracellular bacteria and fungi in the lung. Although there is increasing evidence that IL-17 is involved in protective immunity against H1 and H3 influenza virus infections, little is known about the role of IL-17 in the highly pathogenic H5N1 influenza virus infection. In this study, we show that H5N1-infected IL-17 knockout (KO) mice exhibit markedly increased weight loss, more pronounced lung immunopathology and significantly reduced survival rates as compared with infected wild-type controls. Moreover, the frequency of B cells in the lung were substantially decreased in IL-17 KO mice after virus infection, which correlated with reduced CXCR5 expression in B cells and decreased CXCL13 production in the lung tissue of IL-17 KO mice. Consistent with this observation, B cells from IL-17 KO mice exhibited a significant reduction in chemokine-mediated migration in culture. Taken together, these findings demonstrate a critical role for IL-17 in mediating the recruitment of B cells to the site of pulmonary influenza virus infection in mice.