Hironori Yoshii

Deletion in Open Reading Frame 49 of Varicella-Zoster Virus Reduces Virus Growth in Human Malignant Melanoma Cells but Not in Human Embryonic Fibroblasts

ABSTRACT The ORF49 gene product (ORF49p) of the varicella-zoster virus (VZV) is likely a myristylated tegument protein, and its homologs are conserved across the herpesvirus subfamilies. The UL11 gene of herpes simplex virus type 1 and of pseudorabies virus and the UL99 gene of human cytomegalovirus are the homologs of ORF49 and have been well characterized by using mutant viruses; however, little research on the VZV ORF49 gene has been reported. Here we report on VZV ORF49p expression, subcellular localization, and effect on viral spread in vitro. ORF49p was expressed during the late phase of infection and located in the juxtanuclear region of the cytoplasm, where it colocalized mainly with the trans-Golgi network-associated protein. ORF49p was incorporated into virions and showed a molecular mass of 13 kDa in VZV-infected cells and virions. To elucidate the role of the ORF49 gene, we constructed a mutant virus that lacked a functional ORF49. No differences in plaque size or cell-cell spread were observed in human embryonic fibroblast cells, MRC-5 cells, infected with the wild-type or the mutant virus. However, the mutant virus showed diminished cell-cell infection in a human malignant melanoma cell line, MeWo cells. Therefore, VZV ORF49p is important for virus growth in MeWo cells, but not in MRC-5 cells. VZV may use different mechanisms for virus growth in MeWo and MRC-5 cells. If so, understanding the role of ORF49p should help elucidate how VZV accomplishes cell-cell infections in different cell types.

Varicella-Zoster Virus Glycoprotein M Homolog Is Glycosylated, Is Expressed on the Viral Envelope, and Functions in Virus Cell-to-Cell Spread

ABSTRACT Although envelope glycoprotein M (gM) is highly conserved among herpesviruses, the varicella-zoster virus (VZV) gM homolog has never been investigated. Here we characterized the VZV gM homolog and analyzed its function in VZV-infected cells. The VZV gM homolog was expressed on virions as a glycoprotein modified with a complex N-linked oligosaccharide and localized mainly to the Golgi apparatus and the trans-Golgi network in infected cells. To analyze its function, a gM deletion mutant was generated using the bacterial artificial chromosome system in Escherichia coli, and the virus was reconstituted in MRC-5 cells. VZV is highly cell associated, and infection proceeds mostly by cell-to-cell spread. Compared with wild-type VZV, the gM deletion mutant showed a 90% reduction in plaque size and 50% of the cell-to-cell spread in MRC-5 cells. The analysis of infected cells by electron microscopy revealed numerous aberrant vacuoles containing electron-dense materials in cells infected with the deletion mutant virus but not in those infected with wild-type virus. However, enveloped immature particles termed L particles were found at the same level on the surfaces of cells infected with either type of virus, indicating that envelopment without a capsid might not be impaired. These results showed that VZV gM is important for efficient cell-to-cell virus spread in cell culture, although it is not essential for virus growth.

Single dose of inactivated Japanese encephalitis vaccine with poly(γ-glutamic acid) nanoparticles provides effective protection from Japanese encephalitis virus

Japanese encephalitis (JE) is a serious disease caused by the JE virus (JEV), and vaccination is the only way to prevent the diseases. In Japan, the only JE vaccine currently available is an inactivated vaccine that requires multiple doses for effective protection; therefore, an effective single-dose vaccine is needed. However, no report of an effective protocol for a single dose of JE vaccine in animals has been published. Here, we evaluated the efficacy of a single-dose vaccination in mice to which the JE vaccine was given with or without adjuvant. Biodegradable poly(gamma-glutamic acid) nanoparticles (gamma-PGA-NPs) were used as a test adjuvant. Remarkably, a single dose of JE vaccine with gamma-PGA-NPs enhanced the neutralizing antibody titer, and all of the immunized mice survived a normally lethal JEV infection, while only 50% of the mice that received a single dose of JE vaccine without gamma-PGA-NPs survived. The use of aluminum as the adjuvant showed similar levels of enhanced efficacy. These results show that gamma-PGA-NPs are a novel and safe adjuvant for JE vaccine, and that a single dose of JE vaccine with gamma-PGA-NPs provides effective protection from lethal JEV in mice. A similar protocol, in which a single dose of JE vaccine is mixed with gamma-PGA-NPs, may be useful for the immunization of humans.

Poly-γ-Glutamic Acid Nanoparticles and Aluminum Adjuvant Used as an Adjuvant with a Single Dose of Japanese Encephalitis Virus-Like Particles Provide Effective Protection from Japanese Encephalitis Virus

ABSTRACT To maintain immunity against Japanese encephalitis virus (JEV), a formalin-inactivated Japanese encephalitis (JE) vaccine should be administered several times. The repeated vaccination is not helpful in the case of a sudden outbreak of JEV or when urgent travel to a high-JEV-risk region is required; however, there are few single-injection JE vaccine options. In the present study, we investigated the efficacy of a single dose of a new effective JE virus-like particle preparation containing the JE envelope protein (JE-VLP). Although single administration with JE-VLP protected less than 50% of mice against lethal JEV infection, adding poly(γ-glutamic acid) nanoparticles (γ-PGA-NPs) or aluminum adjuvant (alum) to JE-VLP significantly protected more than 90% of the mice. A single injection of JE-VLP with either γ-PGA-NPs or alum induced a significantly greater anti-JEV neutralizing antibody titer than JE-VLP alone. The enhanced titers were maintained for more than 6 months, resulting in long-lasting protection of 90% of the immunized mice. Although the vaccine design needs further modification to reach 100% protection, a single dose of JE-VLP with γ-PGA-NPs may be a useful step in developing a next-generation vaccine to stop a JE outbreak or to immunize travelers or military personnel.

Varicella-zoster virus ORF1 gene product is a tail-anchored membrane protein localized to plasma membrane and trans-Golgi network in infected cells

Varicella-zoster virus (VZV) encodes five genes that do not have herpes simplex virus homologs. One of these genes, VZV open reading frame 1 (ORF1), encodes a membrane protein with a hydrophobic domain at its C-terminus that is predicted to be the transmembrane domain. However, the detailed characterization of ORF1 protein in infected cells has not been reported. Here, we produced mono-specific antibodies against ORF1 protein and characterized the gene products in infected cells. Western blot analyses showed the ORF1 polypeptides had apparent molecular masses of approximately 14-17 kDa. Furthermore, ORF1 was found to be a phosphoprotein by immunoprecipitation assay. In immunofluorescence assays, the VZV ORF1 protein was detected at both the plasma membrane and trans-Golgi network in both VZV-infected and ORF1-transfected cells. Moreover, ORF1 proteins associated with each other to form homodimer, and were incorporated into viral particles. The C-terminal hydrophobic domain was required for the association of ORF1 with the membrane structures, indicating that ORF1 protein is anchored to the membrane thorough its C-terminus, which is a transmembrane domain. Because ORF1 possesses a C-terminal transmembrane domain without an N-terminal signal sequence for its translocation to the ER lumen, ORF1 can be classified as a tail-anchored membrane protein. These results show that the N terminus of ORF1 protein faces the cytoplasm in infected cells and the tegument region in mature virions.

Serostatus following live attenuated vaccination administered before pediatric liver transplantation

After liver transplantation (LT), live attenuated vaccines (LAVs) are generally contraindicated. LAVs are recommended before LT for patients ≥ 6 months of age. However, the evidence supporting this practice is limited. Patients were enrolled before and after LT. Clinical data for patients were obtained from medical records. Serum antibody titers were evaluated at the time of enrollment and prospectively. Serum antibody titers were measured with a hemagglutination inhibition test for measles and rubella and with an enzyme‐linked immunosorbent assay for varicella and mumps. Univariate and multivariate analyses were performed to investigate the factors that affect the serostatus. Serological analyses of 49 patients immunized before LT (median age, 45 months; male, 35%) were performed. Underlying diseases were biliary atresia (n = 27; 55%), metabolic diseases (n = 13; 27%), fulminant hepatic failure (n = 5; 10%), and others (n = 4; 8%). The seropositivity rate after each vaccine was 46.9% (measles), 89.4% (rubella), 67.5% (varicella), and 48.8% (mumps). Factors independently associated with seronegativity were a vaccination age < 12 months for measles (P = .002), a lower body weight for varicella (P = 0.01), and underlying diseases other than biliary atresia for mumps (P = .004). No serious adverse event was observed during the study period. The immunogenicity of LAVs before LT was high for rubella but low for the others. Before LT, further vaccination strategies are needed for patients. In addition, serological follow‐up may be indicated for patients with factors associated with seronegativity. Liver Transpl 21:774–783, 2015.

Immunogenicity of Quadrivalent Influenza Vaccine for Patients with Inflammatory Bowel Disease Undergoing Immunosuppressive Therapy

Abstract Background and Aims No reports have described the immunogenicity and boosting effect of the quadrivalent inactivated influenza vaccine (QIV) in adults with inflammatory bowel disease. Methods Adults with Crohn’s disease or ulcerative colitis were randomly assigned to a single vaccination group or booster group, and a QIV was administered subcutaneously. Serum samples were collected before vaccination, 4 weeks after vaccination, and after the influenza season in the single vaccination group. In the booster group, serum samples were taken before vaccination, 4 weeks after the first vaccination, 4 weeks after the second vaccination, and after the influenza season. We measured hemagglutination inhibition antibody (HAI) titer and calculated the geometric mean titer ratio (GMTR), seroprotection rate, and seroconversion rate. Results In total, 132 patients were enrolled. Twenty-two patients received immunomodulatory monotherapy and 16 received anti-tumor necrosis factor-α (anti-TNF-α) single-agent therapy. Fifteen patients received combination therapy comprising an immunosuppressant and anti-TNF-α agent. Each vaccine strain showed immunogenicity satisfying the European Medicines Agency criteria with a single inoculation. The booster influenza vaccination did not induce additional response. In patients administered infliximab, the seroprotection rate and seroconversion rate tended to be lower in patients who maintained blood concentrations [seroprotection rate: H1N1: OR, 0.37 (95% CI, 0.11–1.21); H3N2: 0.22 (0.07–0.68); seroconversion rate: H1N1: 0.23 (0.06–0.91); H3N2: 0.19 (0.06–0.56)]. Conclusion Single dose QIV showed sufficient immunogenicity in patients with inflammatory bowel disease, and a boost in immunization by additional vaccination was not obtained. Additionally, immunogenicity was low in patients receiving infliximab therapy.

Cytokine production in whole-blood cultures following immunization with an influenza vaccine

ABSTRACT A clinical trial of a quadrivalent split influenza vaccine was performed in the 2014/15 season. Sixty-four subjects aged 6 months to 18 years were enrolled in order to investigate the relationship between cellular and humoral immune responses. Subjects were categorized into two groups by measuring neutralizing antibodies: non-primed naïve/primed or seroconverted/non-seroconverted groups. Whole-blood cultures were stimulated with the H1N1 split antigen before immunization and one month after the first and second immunizations for subjects < 13 years and before and one month after the first dose for those ≥ 13 years in order to investigate cytokine production. Significant amounts of IL-2, IL-12, IL-13, MCP-1, MIP-1β, and TNF-α were detected from one month after the first dose in the naïve group. In addition to these cytokines, the production of IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17, G-CSF, and IFN-γ was enhanced one month after the second dose. No significant increase was noted in the primed group, except in the production of IL-10. In seroconverted subjects, the production of IL-2, IL-4, IL-8, IL-10, G-CSF, MCP-1, TNF-α, and IFN-γ increased one month after the first dose, which was earlier than in the naïve group, whereas no significant cytokine response was noted in subjects without seroconversion. Subjects ≥ 13 years were primed and the production of G-CSF, IL-4, and IL-1β increased in subjects with seroconversion. Whole-blood cultures were also stimulated with the H3N2 split antigen and similar cytokine profiles were obtained. Many cytokines and chemokines, including inflammatory cytokines, were produced in seroconverted, but not non-seroconverted subjects.