ヒトサル痘の迅速診断法の開発および高度弱毒痘そうワクチンの霊長類をモデルとした評価

学位の種別: 課程博士審査委員会委員 : (主査)東京大学教授 岡 明, 東京大学教授 北 潔, 東京大学准教授 渡邊 洋一, 東京大学准教授 大迫 誠一郎, 東京大学准教授 米田 美佐子University of Tokyo(東京大学

Correction to: A loop-mediated isothermal amplification assay for the detection and quantification of JC polyomavirus in cerebrospinal fluid: a diagnostic and clinical management tool and technique for progressive multifocal leukoencephalopathy

In the original publication of article [1], ‘20 × 101 copies’, which is in the sentence ‘As seen in Fig. 4, the sensitivity of the specimens containing equal to or more than 20 × 10 1 copies in 2 μL of extracted DNA (equivalent to ≥3.0 × 103 copies/mL CSF) was 100% (29/29)’ changes to ‘2.0 × 101 copies’ in results section. The publisher apologizes to the readers and authors for the inconvenience

Genetic Characterization of Human Rabies Vaccine Strain in Japan and Rabies Viruses Related to Vaccine Development from 1940s to 1980s

The rabies virus is widely distributed and vaccines are an important strategy to prevent its spread. The whole-genome sequences of rabies strains in relation to vaccine development provide essential information to maintain vaccine quality and develop new vaccines. However, the genetic characteristics of the purified chick embryo cell culture rabies vaccine, KM Biologics (PCECV-KMB), developed in Japan in the 1970s, have not been explored. In this study, we conducted a genome-wide analysis of the open reading frame regions of rabies strains discovered from the 1940s–1980s and used to develop chick embryo cell-adapted HEP-Flury small plaque-forming (CEF-S) strain, which is a vaccine strain of PCECV-KMB. The genetic characteristic of CEF-S, developed by acclimation of the HEP-Flury-NIID strain to one-day eggs and subsequently to chick embryo cells, were confirmed by comparing the genome identity and revealing the nine amino acid mutations between CEF-S and HEP-Flury-NIID. The efficacy of PCECV-KMB was evaluated using attack strains isolated in Thailand in the 1960s–1970s during vaccine development. Phylogenetic analyses of the attack strains classified them in the same Asian clade as the 2000s imported cases from the Philippines to Japan, suggesting that PCECV-KMB is adequate for preventing the spread of the current rabies virus

Replication-incompetent rabies virus vector harboring glycoprotein gene of lymphocytic choriomeningitis virus (LCMV) protects mice from LCMV challenge.

BACKGROUND:Lymphocytic choriomeningitis virus (LCMV) causes a variety of diseases, including asymptomatic infections, meningitis, and congenital infections in the fetus of infected mother. The development of a safe and effective vaccine against LCMV is imperative. This study aims to develop a new candidate vaccine against LCMV using a recombinant replication-incompetent rabies virus (RV) vector. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we have generated a recombinant deficient RV expressing the LCMV glycoprotein precursor (GPC) (RVΔP-LCMV/GPC) which is lacking the RV-P gene. RVΔP-LCMV/GPC is able to propagate only in cells expressing the RV-P protein. In contrast, the LCMV-GPC can be expressed in general cells, which do not express RV-P protein. The ability of RVΔP-LCMV/GPC to protect mice from LCMV infection and induce cellular immunity was assessed. Mice inoculated intraperitoneally with RVΔP-LCMV/GPC showed higher survival rates (88.2%) than those inoculated with the parental recombinant RV-P gene-deficient RV (RVΔP) (7.7%) following a LCMV challenge. Neutralizing antibody (NAb) against LCMV was not induced, even in the sera of surviving mice. CD8+ T-cell depletion significantly reduced the survival rates of RVΔP-LCMV/GPC-inoculated mice after the LCMV challenge. These results suggest that CD8+ T cells play a major role in the observed protection against LCMV. In contrast, NAbs against RV were strongly induced in sera of mice inoculated with either RVΔP-LCMV/GPC or RVΔP. In safety tests, suckling mice inoculated intracerebrally with RVΔP-LCMV/GPC showed no symptoms. CONCLUSIONS/SIGNIFICANCE:These results show RVΔP-LCMV/GPC might be a promising candidate vaccine with dual efficacy, protecting against both RV and LCMV

Growth kinetics of RVΔP-LCMV/GPC and RVΔP in BHK-P cells.

<p>BHK-P cells were inoculated with RVΔP-LCMV/GPC or RVΔP at an MOI of 0.01. Culture supernatants were harvested at 1, 3, 5, and 7 days post inoculation, and virus titers were determined using BHK-P cells. The data were expressed as mean ± SE of 4 independent experiments. Asterisks indicate a significant difference (p < 0.05).</p

Analysis of the humoral immune responses among cynomolgus macaque naturally infected with Reston virus during the 1996 outbreak in the Philippines

<p>Abstract</p> <p>Background</p> <p>Ebolaviruses induce lethal viral hemorrhagic fevers (VHFs) in humans and non-human primates, with the exceptions of Reston virus (RESTV), which is not pathogenic for humans. In human VHF cases, extensive analyses of the humoral immune responses in survivors and non-survivors have shown that the IgG responses to nucleoprotein (NP) and other viral proteins are associated with asymptomatic and survival outcomes, and that the neutralizing antibody responses targeting ebolaviruses glycoprotein (GP_1,2) are the major indicator of protective immunity. On the other hand, the immune responses in non-human primates, especially naturally infected ones, have not yet been elucidated in detail, and the significance of the antibody responses against NP and GP_1,2 in RESTV-infected cynomolgus macaques is still unclear. In this study, we analyzed the humoral immune responses of cynomolgus macaque by using serum specimens obtained from the RESTV epizootic in 1996 in the Philippines to expand our knowledge on the immune responses in naturally RESTV-infected non-human primates.</p> <p>Results</p> <p>The antibody responses were analyzed using IgG-ELISA, an indirect immunofluorescent antibody assay (IFA), and a pseudotyped VSV-based neutralizing (NT) assay. Antigen-capture (Ag)-ELISA was also performed to detect viral antigens in the serum specimens. We found that the anti-GP_1,2 responses, but not the anti-NP responses, closely were correlated with the neutralization responses, as well as the clearance of viremia in the sera of the RESTV-infected cynomolgus macaques. Additionally, by analyzing the cytokine/chemokine concentrations of these serum specimens, we found high concentrations of proinflammatory cytokines/chemokines, such as IFNγ, IL8, IL-12, and MIP1α, in the convalescent phase sera.</p> <p>Conclusions</p> <p>These results imply that both the antibody response to GP_1,2 and the proinflammatory innate responses play significant roles in the recovery from RESTV infection in cynomolgus macaques.</p

Survival curves in mice inoculated with adenovirus vectors before LCMV-WE infection.

<p>(A) The schedules of immunization with adenovirus and LCMV challenge in C57BL/6 mice. Mice were inoculated twice with Ax-LCMV/GPC or Ax-empty at 1-week intervals. One week after the last inoculation of adenovirus, mice were challenged with 10 PFU of LCMV-WE and observed for 2 weeks. (B) The survival curve of mice inoculated with Ax-LCMV/GPC or Ax-empty. Asterisks indicate a significant difference (p < 0.001).</p

Survival curves and NAb titers in mice infected with LCMV-WE with or without CD8+ T cell depletion.

<p>(A) RVΔP-LCMV/GPC inoculation, challenge, and blood collection schedules in C57BL/6 mice. Mice were inoculated with RVΔP-LCMV/GPC twice at 3-week intervals. Mice were injected with anti-CD8+ T cell antibody (n = 6) or Isotype control (n = 6) 4 days and 7 days after the last RVΔP-LCMV/GPC inoculation. Seven days after the last inoculation, mice were intracranially infected with 10 PFU of LCMV-WE. Mouse serum was collected 1 day before the first RV inoculation (Pre) and second RV inoculation (1st), and the serum of the surviving mice was collected on the last day of the 3-week observation (Post). (B) The survival curve of mice injected with CD8+ T cell antibody or isotype control. The titers of anti-LCMV (C) and anti-RV (D) NAb in mice with and without CD8+ T-cell depletion.</p

Confirmation of RV-N and LCMV-GP expression.

<p>BHK-P cells were inoculated with RVΔP-LCMV/GPC (A–C) or RVΔP (D–F) at an MOI of 0.1 and incubated at 33°C for 48 h. Cells were stained with anti-RV N mAb or the anti-LCMV-GP1 mAb. Original magnification, 400×. (G–I) RV-N and the LCMV-GPC were detected in Neuro-2a cells inoculated with RVΔP-LCMV/GPC at an MOI of 8 at 48 h post inoculation). Original magnification, 200×. (J) GPC (70 KDa) and GP1 (40 KDa) proteins were detected in BHK-P cells infected with RVΔP-LCMV/GPC by western blot using the anti-LCMV-GP1 mAb. (K–L) The cultured medium (Sup) of cells infected with RVΔP-LCMV/GPC or RVΔP were PEG precipitated (PEG ppt) and purified (Purified) by ultracentrifugation and stained with the anti LCMV-GP1 Mab (K) or the anti-RV G mAb (L). The lysates of mock-inoculated Vero cells and LCMV-WE -inoculated Vero cells were used as negative and positive controls, respectively (K). Lysates of mock-inoculated Neuro-2a cells and RV HEP-Flury strain-infected Neuro-2a cells were used as negative and positive controls, respectively (L).</p