Toyokazu Ishikawa

Poly(γ-glutamic acid) nano-particles combined with mucosal influenza virus hemagglutinin vaccine protects against influenza virus infection in mice

Adding poly(gamma-glutamic acid) nano-particles (gamma-PGA-NPs), a safe, natural material, to subcutaneous immunization with influenza virus hemagglutinin (HA) vaccine increases the protective immune responses against influenza virus in mice. Here, we examined whether intranasal administration of the HA vaccine with gamma-PGA-NPs would induce protection from influenza virus challenge in mice. Intranasal immunization with the mixture of gamma-PGA-NPs and HA vaccine from an influenza virus strain A/PR/8/34 (H1N1) or A/New Caledonia/20/99 (H1N1) enhanced protection of mice from A/PR/8/34 infection. Intranasal immunization with A/New Caledonia/20/99 HA vaccine and gamma-PGA-NPs induced cell-mediated immune responses and neutralizing antibody production for both A/New Caledonia/20/99 and A/PR/8/34. These data suggest that gamma-PGA-NPs may have potential for clinical applications as a mucosal adjuvant.

Anti-influenza activity of marchantins, macrocyclic bisbibenzyls contained in liverworts.

The H1N1 influenza A virus of swine-origin caused pandemics throughout the world in 2009 and the highly pathogenic H5N1 avian influenza virus has also caused epidemics in Southeast Asia in recent years. The threat of influenza A thus remains a serious global health issue and novel drugs that target these viruses are highly desirable. Influenza A possesses an endonuclease within its RNA polymerase which comprises PA, PB1 and PB2 subunits. To identify potential new anti-influenza compounds in our current study, we screened 33 different types of phytochemicals using a PA endonuclease inhibition assay in vitro and an anti-influenza A virus assay. The marchantins are macrocyclic bisbibenzyls found in liverworts, and plagiochin A and perrottetin F are marchantin-related phytochemicals. We found from our screen that marchantin A, B, E, plagiochin A and perrottetin F inhibit influenza PA endonuclease activity in vitro. These compounds have a 3,4-dihydroxyphenethyl group in common, indicating the importance of this moiety for the inhibition of PA endonuclease. Docking simulations of marchantin E with PA endonuclease suggest a putative “fitting and chelating model” as the mechanism underlying PA endonuclease inhibition. The docking amino acids are well conserved between influenza A and B. In a cultured cell system, marchantin E was further found to inhibit the growth of both H3N2 and H1N1 influenza A viruses, and marchantin A, E and perrotein F showed inhibitory properties towards the growth of influenza B. These marchantins also decreased the viral infectivity titer, with marchantin E showing the strongest activity in this assay. We additionally identified a chemical group that is conserved among different anti-influenza chemicals including marchantins, green tea catechins and dihydroxy phenethylphenylphthalimides. Our present results indicate that marchantins are candidate anti-influenza drugs and demonstrate the utility of the PA endonuclease assay in the screening of phytochemicals for anti-influenza characteristics.

Measurement of Varicella-Zoster Virus (VZV)-Specific Cell-Mediated Immunity: Comparison between VZV Skin Test and Interferon-γ Enzyme-Linked Immunospot Assay

Cell-mediated immunity (CMI) is critical for the prevention and control of varicella-zoster virus (VZV)-related disease. To assess CMI to VZV, a varicella skin test and interferon-gamma enzyme-linked immunospot (ELISPOT) assay were both performed in healthy volunteers, and the results were compared. A total of 151 subjects were examined: 16 aged 20-29 years, 26 aged 30-39 years, 18 aged 40-49 years, 73 aged 50-59 years, and 18 aged 60-69 years. All were seropositive by a glycoprotein antigen-based enzyme-linked immunosorbent assay (gpELISA). Skin test reactivity was significantly correlated with the ELISPOT count, and both decreased with increasing age, indicating an age-dependent decline in CMI to VZV. In contrast, the antibody titer obtained by the gpELISA did not correlate with skin test reactivity. The results suggest that the skin test and ELISPOT assay are both reliable for assessing CMI to VZV and can easily be applied to screen individuals susceptible to the development of herpes zoster.

Stable High-Producer Cell Clone Expressing Virus-Like Particles of the Japanese Encephalitis Virus E Protein for a Second-Generation Subunit Vaccine

ABSTRACT We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.5 μg per 104 cells) to the internationally licensed JE vaccine JE-VAX. E-VLP production was stable after multiple cell passages and persisted over 1 year with 100% expressing cells without detectable cell fusion, apoptosis, or cell death, but was suspended when the cells grew to 100% confluency and contact inhibition occurred. Mice immunized with the purified J12#26 E-antigen without adjuvant developed high titers of neutralizing antibodies for at least 7 months and 100% protection against intraperitoneal challenge with 5 × 106 PFU of JEV when examined according to the JE vaccine standardization protocol. These results suggest that the recombinant E-VLP antigen produced by the J12#26 cell clone is an effective, safe, and low-cost second-generation subunit JE vaccine.

Immunogenicity and efficacy of two types of West Nile virus-like particles different in size and maturation as a second-generation vaccine candidate.

Virus-like particles (VLPs) of flaviviruses generated from the prM and E genes are a promising vaccine candidate. We have established cell clones continuously releasing VLPs of West Nile virus (WNV) in serum-free conditions. Two types of VLPs were distinguished by sedimenting analyses in sucrose density gradients. Fast sedimenting VLPs (F-VLPs) were large (40-50 nm) and composed of the E and processed mature M proteins, whereas slowly sedimenting VLPs (S-VLPs) were small (20-30 nm) particles consisting of the E and immature prM proteins. F-VLPs induced higher neutralizing antibody and anti-WNV IgG titers than S-VLPs. Furthermore, IgG2a was dominant over IgG1 by immunization with F-VLPs as with whole virion-derived antigens. Mice vaccinated with a low dose (3 ng) of F-VLPs showed higher protective efficacy (83% survivals) against WNV infection than S-VLP-immune mice (17% survivals). These results indicate that F-VLPs more closely resemble the virions and take a better immunogenic form than S-VLPs as WNV vaccine candidates.

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.

Monoclonal antibodies in man that neutralized H3N2 influenza viruses were classified into three groups with distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003.

We tried to reveal the strain specificity of neutralizing mAbs against H3N2 influenza viruses in individuals. A large number of B lymphocytes of a pediatrician were collected by apheresis and two Ab libraries were constructed at 2004 and 2007 by using the phage-display technology. The libraries were screened against 12 different H3 strains of flu isolated between 1968 and 2004. Large numbers of clones that bound to the Ags were isolated and mAbs that specifically bound to H3 strain viruses were selected. Their binding activity to the 12 strains and neutralizing activity were studied by ELISA and focus reduction test, respectively. Furthermore, the binding activity to hemagglutinin (HA) was examined by Western blot. The majority of clones showing the neutralizing activity turned out to be anti-HA mAbs and could be divided into three major groups showing distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003.

Incidences of Herpes Zoster and Postherpetic Neuralgia in Japanese Adults Aged 50 Years and Older From a Community-based Prospective Cohort Study: The SHEZ Study

Background Many cross-sectional studies have examined the incidences of herpes zoster (HZ) and postherpetic neuralgia (PHN), but prospective studies in Japanese older adults are lacking. Therefore, we conducted a community-based prospective cohort study to determine the incidence in Japanese adults aged ≥50 years. Methods We recruited 12 522 participants from Shozu County, Kagawa Prefecture, between December 2008 and November 2009 and followed participants for 3 years. When a subject presented with symptoms suggestive of HZ, they were examined at collaborating medical institutions and cooperated with onset and recovery surveys (eg, measurement of varicella zoster virus-specific immunity and a pain survey). The hazard ratios (HRs) of HZ and PHN according to sex and age were analyzed by Cox regression analysis with a significance level of 5%. Results The incidence of HZ was 10.9/1000 person-years (men: 8.5/1000 person-years; women: 12.8/1000 person-years) and was significantly higher in women than in men (HR 1.5; 95% confidence interval, 1.2–1.8). The incidence of PHN was 2.1/1000 person-years (men: 1.7/1000 person-years; women: 2.4/1000 person-years), with no significant sex differences. A total of 19% of HZ cases progressed to PHN; no sex-specific difference in the proportion of PHN cases was observed. Conclusions We clarified the accurate incidences of HZ and PHN in a population of Japanese older adults. These incidences increased with age. HZ incidence was higher in women than in men, while PHN incidence did not differ markedly between the sexes.

Superior immunogenicity of a freeze-dried, cell culture-derived Japanese encephalitis vaccine (inactivated).

Japanese encephalitis is an infectious disease caused by the Japanese encephalitis virus, which is widespread throughout Asia. The worldwide incidence is 50,000 cases per year. There is no specific treatment available, but inactivated mouse brain-derived vaccine was used from the 1950s to prevent infection. However, quality control of mouse brain-derived vaccines is difficult, and therefore a new freeze-dried, cell culture-derived Japanese encephalitis vaccine (inactivated) (JEBIK V; development code: BK-VJE) was developed. In this paper, we report an analysis of neutralizing antibody titers in vaccinated subjects enrolled in clinical study of BK-VJE at various doses, and study of BK-VJE with the mouse brain-derived vaccine as a control. The results show that BK-VJE has superior immunogenicity compared to mouse brain-derived vaccine.

Characterization of neutralizing antibodies in adults after intranasal vaccination with an inactivated influenza vaccine

The levels and properties of neutralizing antibodies in nasal wash and serum collected from five healthy adults were examined after intranasal administration of an A/Uruguay/716/2007 (H3N2) split vaccine (45 µg hemagglutinin (HA) per dose; five doses, with an interval of 3 weeks between each dose). Prior to the assays, nasal wash samples were concentrated so that the total amount of antibodies was equivalent to about 1/10 of that found in the natural nasal mucus. Vaccination induced virus‐specific neutralizing antibody responses, which increased with the number of vaccine doses given. Neutralizing antibodies were produced more efficiently in the nasal passages than in the serum: A ≥4‐fold increase in nasal neutralization titres was observed after the second vaccination in four out of five subjects, whereas a rise in serum neutralization titres was observed only after the fifth vaccination. Nasal and serum neutralizing antibodies were mainly found in the polymeric IgA and monomeric IgG fractions, respectively, after gel filtration. Taken together, these results suggest that intranasal administration of an inactivated split vaccine induces high levels of nasal neutralizing antibodies (primarily polymeric IgA) and low levels of serum neutralizing antibodies (primarily monomeric IgG). J. Med. Virol. 84:336–344, 2012.