Fumihiko Yasui

Evaluation of a cyclophilin inhibitor in hepatitis C virus-infected chimeric mice in vivo.

Cyclosporin A (CsA) inhibits replication of the HCV subgenomic replicon, and this effect is believed to not be mediated by its immunosuppressive action. We found that DEBIO‐025, a novel non‐immunosuppressive cyclophilin inhibitor derived from CsA, inhibited HCV replication in vitro more potently than CsA. We also examined the inhibitory effect of DEBIO‐025 on naive HCV genotypes 1a or 1b in vivo using chimeric mice with human hepatocytes. These mice were treated for 14 days with DEBIO‐025, pegylated‐interferon α−2a (Peg‐IFN), a combination of either drugs, or CsA in combination with Peg‐IFN. In mice treated with Peg‐IFN, serum HCV RNA levels decreased approximately 10‐fold whereas DEBIO‐025 treatment alone did not induce any significant change. In mice treated with both DEBIO‐025 and Peg‐IFN, HCV RNA levels decreased more than 100‐fold. All mice treated with Peg‐IFN combined with CsA died within 4 days. The combination treatment of DEBIO‐025 and Peg‐IFN reduced HCV RNA levels and core protein expression in liver, indicating that the HCV RNA levels reduction in serum was attributable to intrahepatic inhibition of HCV replication. Conclusion: We demonstrated that DEBIO‐025 was better tolerated than CsA, and that its anti‐HCV effect appeared to be synergistic in combination with Peg‐IFN in vivo. (HEPATOLOGY 2007;45:921–928.)

Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein Causes Severe Pneumonia in Mice Infected with SARS-CoV

The details of the mechanism by which severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia are unclear. We investigated the immune responses and pathologies of SARS-CoV-infected BALB/c mice that were immunized intradermally with recombinant vaccinia virus (VV) that expressed either the SARS-CoV spike (S) protein (LC16m8rVV-S) or simultaneously all the structural proteins, including the nucleocapsid (N), membrane (M), envelope (E), and S proteins (LC16m8rVV-NMES) 7–8 wk before intranasal SARS-CoV infection. The LC16m8rVV-NMES-immunized group exhibited as severe pneumonia as the control groups, although LC16m8rVV-NMES significantly decreased the pulmonary SARS-CoV titer to the same extent as LC16m8rVV-S. To identify the cause of the exacerbated pneumonia, BALB/c mice were immunized with recombinant VV that expressed the individual structural proteins of SARS-CoV (LC16mOrVV-N, -M, -E, -S) with or without LC16mOrVV-S (i.e., LC16mOrVV-N, LC16mOrVV-M, LC16mOrVV-E, or LC16mOrVV-S alone or LC16mOrVV-N + LC16mOrVV-S, LC16mOrVV-M + LC16mOrVV-S, or LC16mOrVV-E + LC16mOrVV-S), and infected with SARS-CoV more than 4 wk later. Both LC16mOrVV-N-immunized mice and LC16mOrVV-N + LC16mOrVV-S-immunized mice exhibited severe pneumonia. Furthermore, LC16mOrVV-N-immunized mice upon infection exhibited significant up-regulation of both Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5) cytokines and down-regulation of anti-inflammatory cytokines (IL-10, TGF-β), resulting in robust infiltration of neutrophils, eosinophils, and lymphocytes into the lung, as well as thickening of the alveolar epithelium. These results suggest that an excessive host immune response against the nucleocapsid protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase our understanding of the pathogenesis of SARS.

Augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/X1 and 7a/X4 proteins through NF-κB activation

Severe acute respiratory syndrome (SARS) is characterized by rapidly progressing respiratory failure resembling acute/adult respiratory distress syndrome (ARDS) associated with uncontrolled inflammatory responses. Here, we demonstrated that, among five accessory proteins of SARS coronavirus (SARS‐CoV) tested, 3a/X1 and 7a/X4 were capable of activating nuclear factor kappa B (NF‐κB) and c‐Jun N‐terminal kinase (JNK), and significantly enhanced interleukin 8 (IL‐8) promoter activity. Furthermore, 3a/X1 and 7a/X4 expression in A549 cells enhanced production of inflammatory chemokines that were known to be up‐regulated in SARS‐CoV infection. Our results suggest potential involvement of 3a/X1 and 7a/X4 proteins in the pathological inflammatory responses in SARS.

Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29(+/−)/MxCre(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

SARS-CoV spike protein-expressing recombinant vaccinia virus efficiently induces neutralizing antibodies in rabbits pre-immunized with vaccinia virus.

Abstract A vaccine for severe acute respiratory syndrome (SARS) is being intensively pursued against its re-emergence. We generated a SARS coronavirus (SARS-CoV) spike protein-expressing recombinant vaccinia virus (RVV-S) using highly attenuated strain LC16m8. Intradermal administration of RVV-S into rabbits induced neutralizing (NT) antibodies against SARS-CoV 1 week after administration and the NT titer reached 1:1000 after boost immunization with RVV-S. Significantly, NT antibodies against SARS-CoV were induced by administration of RVV-S to rabbits that had been pre-immunized with LC16m8. RVV-S can induce NT antibodies against SARS-CoV despite the presence of NT antibodies against VV. These results suggest that RVV-S may be a powerful SARS vaccine, including in patients previously immunized with the smallpox vaccine.

Identification of SARS-COV spike protein-derived and HLA-A2-restricted human CTL epitopes by using a new muramyl dipeptide-derivative adjuvant

Severe acute respiratory syndrome (SARS) spread during the winter of 2003, and attempt have been made to develop vaccines against SARS corona virus (SARS-CoV). The present study provides a strategy to rapidly identify SARS-CoV-derived antigenic peptides recognized by HLA-A2-restricted cytotoxic T lymphocytes (CTLs). Forty-three candidate peptides having HLA-A2-binding motifs were selected in silico and HLA-A2/Db chimeric MHC class I-transgenic mice were immunized with these peptides and a new derivative of muramyl dipeptide that can induce upregulation of HLA-DR, CD80, CD86, and CD40 in human CD14+ antigen presenting cells, was administered as an adjuvant. Six HLA-A2-restricted mouse CTL epitopes were identified, including two new epitopes which have never been reported before. One of the novel peptides was naturally processed and successfully induced HLA-A2-restricted specific CTLs in both HLA transgenic mice and healthy donors. The method was useful, convenient and efficient for rapid identification of CTL epitopes derived from SARS-CoV proteins and will be possibly applicable for other pathogens to develop a peptide-based vaccine.

Histopathological Evaluation of the Diversity of Cells Susceptible to H5N1 Virulent Avian Influenza Virus

Patients infected with highly pathogenic avian influenza A H5N1 viruses (H5N1 HPAIV) show diffuse alveolar damage. However, the temporal progression of tissue damage and repair after viral infection remains poorly defined. Therefore, we assessed the sequential histopathological characteristics of mouse lung after intranasal infection with H5N1 HPAIV or H1N1 2009 pandemic influenza virus (H1N1 pdm). We determined the amount and localization of virus in the lung through IHC staining and in situ hybridization. IHC used antibodies raised against the virus protein and antibodies specific for macrophages, type II pneumocytes, or proliferating cell nuclear antigen. In situ hybridization used RNA probes against both viral RNA and mRNA encoding the nucleoprotein and the hemagglutinin protein. H5N1 HPAIV infection and replication were observed in multiple lung cell types and might result in rapid progression of lung injury. Both type II pneumocytes and macrophages proliferated after H5N1 HPAIV infection. However, the abundant macrophages failed to block the viral attack, and proliferation of type II pneumocytes failed to restore the damaged alveoli. In contrast, mice infected with H1N1 pdm exhibited modest proliferation of type II pneumocytes and macrophages and slight alveolar damage. These results suggest that the virulence of H5N1 HPAIV results from the wide range of cell tropism of the virus, excessive virus replication, and rapid development of diffuse alveolar damage.

Synthetic lipophilic antioxidant BO-653 suppresses HCV replication†

The influence of the intracellular redox state on the hepatitis C virus (HCV) life cycle is poorly understood. This study demonstrated the anti‐HCV activity of 2,3‐dihydro‐5‐hydroxy‐2,2‐dipentyl‐4,6‐di‐tert‐butylbenzofuran (BO‐653), a synthetic lipophilic antioxidant, and examined whether BO‐653s antioxidant activity is integral to its anti‐HCV activity. The anti‐HCV activity of BO‐653 was investigated in HuH‐7 cells bearing an HCV subgenomic replicon (FLR3‐1 cells) and in HuH‐7 cells infected persistently with HCV (RMT‐tri cells). BO‐653 inhibition of HCV replication was also compared with that of several hydrophilic and lipophilic antioxidants. BO‐653 suppressed HCV replication in FLR3‐1 and RMT‐tri cells in a concentration‐dependent manner. The lipophilic antioxidants had stronger anti‐HCV activities than the hydrophilic antioxidants, and BO‐653 displayed the strongest anti‐HCV activity of all the antioxidants examined. Therefore, the anti‐HCV activity of BO‐653 was examined in chimeric mice harboring human hepatocytes infected with HCV. The combination treatment of BO‐653 and polyethylene glycol‐conjugated interferon‐α (PEG‐IFN) decreased serum HCV RNA titer more than that seen with PEG‐IFN alone. These findings suggest that both the lipophilic property and the antioxidant activity of BO‐653 play an important role in the inhibition of HCV replication. J. Med. Virol. 85:241–249, 2013.

Experimental infection of macaques with a wild water bird-derived highly pathogenic avian influenza virus (H5N1).

Highly pathogenic avian influenza virus (HPAIV) continues to threaten human health. Non-human primate infection models of human influenza are desired. To establish an animal infection model with more natural transmission and to determine the pathogenicity of HPAIV isolated from a wild water bird in primates, we administered a Japanese isolate of HPAIV (A/whooper swan/Hokkaido/1/2008, H5N1 clade 2.3.2.1) to rhesus and cynomolgus monkeys, in droplet form, via the intratracheal route. Infection of the lower and upper respiratory tracts and viral shedding were observed in both macaques. Inoculation of rhesus monkeys with higher doses of the isolate resulted in stronger clinical symptoms of influenza. Our results demonstrate that HPAIV isolated from a water bird in Japan is pathogenic in monkeys by experimental inoculation, and provide a new method for HPAIV infection of non-human primate hosts, a good animal model for investigation of HPAIV pathogenicity.

Sensitization with vaccinia virus encoding H5N1 hemagglutinin restores immune potential against H5N1 influenza virus

H5N1 highly pathogenic avian influenza (H5N1 HPAI) virus causes elevated mortality compared with seasonal influenza viruses like H1N1 pandemic influenza (H1N1 pdm) virus. We identified a mechanism associated with the severe symptoms seen with H5N1 HPAI virus infection. H5N1 HPAI virus infection induced a decrease of dendritic cell number in the splenic extrafollicular T-cell zone and impaired formation of the outer layers of B-cell follicles, resulting in insufficient levels of antibody production after infection. However, in animals vaccinated with a live recombinant vaccinia virus expressing the H5 hemagglutinin, infection with H5N1 HPAI virus induced parafollicular dendritic cell accumulation and efficient antibody production. These results indicate that a recombinant vaccinia encoding H5 hemagglutinin gene does not impair dendritic cell recruitment and can be a useful vaccine candidate.