Mikihiro Yunoki

Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors.

Abstract Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine–human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

Long-term shedding of hepatitis E virus in the feces of pigs infected naturally, born to sows with and without maternal antibodies.

Pigs are presumed reservoirs for hepatitis E virus (HEV) transmission to humans. To examine infection kinetics, two litters of domestic pigs (A and B, each containing 10 piglets) infected naturally with HEV were studied until pigs were 6 months old. Maternal IgG and IgA antibodies were detected in litter A piglets, but not in litter B ones. All pigs shed HEV in feces when they were 30–110 days old, and 17 developed viremia at 40–100 days of age. Phylogenetic analysis revealed a highly close sequence of HEV genotype 3 in all pigs. The serum levels of specific IgG and IgA were similar in all pigs, although IgA was not detected in the feces. Interestingly, the onset of both viremia and seroconversion was delayed significantly in litter A pigs. The kinetics of fecal virus shedding was similar in both litters; shedding was not detected after the pigs were 120 days old. The differences in the infection kinetics between litters A and B suggested that maternal antibodies delayed the onset of viremia and seroconversion. Quantitative real‐time reverse transcriptase‐polymerase chain reaction revealed that HEV RNA in feces peaked 10 days after initial shedding of approximately 106.0 copies/g. The viral load was much lower in the serum than in the feces. At 200 days of age, HEV RNA was found in the internal organs of 3 out of 13 pigs. These study findings improve the understanding of the dynamics of natural HEV transmission in pigs, which could help in controlling virus transmission from pigs to humans. J. Med. Virol. 82:69–76, 2010.

Hepatitis E virus in Norway rats (Rattus norvegicus) captured around a pig farm

BackgroundHepatitis E virus (HEV) transmitted via the oral route through the consumption of contaminated water or uncooked or undercooked contaminated meat has been implicated in major outbreaks. Rats may play a critical role in HEV outbreaks, considering their negative effects on environmental hygiene and food sanitation. Although the serological evidence of HEV infection in wild rodents has been reported worldwide, the infectivity and propagation of HEV in wild rats remain unknown. To investigate if rats are a possible carrier of HEV, we studied wild Norway rats (Rattus norvegicus) that were caught near a pig farm, where HEV was prevalent among the pigs.MethodsWe examined 56 Norway rats for HEV. RNA from internal organs was examined for RT-PCR and positive samples were sequenced. Positive tissue samples were incubated with A549 cell line to isolate HEV. Anti-HEV antibodies were detected by ELISA.ResultsSixteen rats were seropositive, and the HEV RNA was detected in 10 of the 56 rats. Sequencing of the partial ORF1 gene from 7 samples resulted in partially sequenced HEV, belonging to genotype 3, which was genetically identical to the HEV prevalent in the swine from the source farm. The infectious HEVs were isolated from the Norway rats by using the human A549 cell line.ConclusionsThere was a relatively high prevalence (17.9%) of the HEV genome in wild Norway rats. The virus was mainly detected in the liver and spleen. The results indicate that these animals might be possible carrier of swine HEV in endemic regions. The HEV contamination risk due to rats needs to be examined in human habitats.

Extent of hepatitis E virus elimination is affected by stabilizers present in plasma products and pore size of nanofilters

Background and Objective  To investigate the physico‐chemical properties of hepatitis E virus (HEV) with regard to inactivation/removal, we have studied four isolates with respect to sensitivity to heat during liquid/dry‐heating as well as removal by nanofiltration.

Highly conserved sequences for human neutralization epitope on hemagglutinin of influenza A viruses H3N2, H1N1 and H5N1: Implication for human monoclonal antibody recognition

The epitope sequences within the hemagglutinin (HA) of influenza A virus H3N2 at amino acid residues 173-181 and 227-239 that forms anti-parallel beta-sheet structure are similarly recognized by human monoclonal antibodies (HuMAbs), B-1 and D-1 that we recently obtained using the peripheral blood lymphocytes from two influenza-vaccinated volunteers. Both HuMAbs showed strong global neutralization of H3N2 strains. Here we show the significant conservation of the beta-sheet region consisting of the above-mentioned two epitope regions in H3N2. In addition, we also identified the corresponding regions with similar structure in other subtypes such as H1N1 and H5N1. These two regions are similarly located underneath the receptor-binding sites of individual subtypes. Analysis of those regions using sequences available from the Influenza Virus Resource at the National Center for Biotechnology Information revealed that compared with those in the known neutralizing epitopes A-E, those sequences were fairly conserved in human H3N2 (n=7955), swine H1N1 (n=360) and swine H3N2 (n=235); and highly conserved in human H1N1 (n=2722), swine-origin pandemic H1N1 (n=1474), human H5N1 (n=319) and avian H5N1 (n=2349). Phylogenetic tree for these regions formed clearly separable clusters for H1N1, H3N2 and H5N1, irrespective of different host origin. These data may suggest a possible significance of those regions for development of alternative vaccine that could induce neutralizing antibodies reactive against wide-range of influenza virus strains.

Variability of parvovirus B19 to inactivation by liquid heating in plasma products

Background and Objectives  Previously, we reported that although human parvovirus B19 in albumin and intravenous immunoglobulin preparations was rapidly inactivated during liquid heating, in contrast to other parvoviruses such as canine parvovirus, sensitivity to heat was highly dependent on the composition of the solution. In this study, we aimed to further elucidate the sensitivity to heat of B19 in haptoglobin and antithrombin (previously named antithrombin III) preparations during liquid heating.

Development of Two Types of Rapid Diagnostic Test Kits to Detect the Hemagglutinin or Nucleoprotein of the Swine-Origin Pandemic Influenza A Virus H1N1

ABSTRACT Since its emergence in April 2009, pandemic influenza A virus H1N1 (H1N1 pdm), a new type of influenza A virus with a triple-reassortant genome, has spread throughout the world. Initial attempts to diagnose the infection in patients using immunochromatography (IC) relied on test kits developed for seasonal influenza A and B viruses, many of which proved significantly less sensitive to H1N1 pdm. Here, we prepared monoclonal antibodies that react with H1N1 pdm but not seasonal influenza A (H1N1 and H3N2) or B viruses. Using two of these antibodies, one recognizing viral hemagglutinin (HA) and the other recognizing nucleoprotein (NP), we developed kits for the specific detection of H1N1 pdm and tested them using clinical specimens of nasal wash fluid or nasopharyngeal fluid from patients with influenza-like illnesses. The specificities of both IC test kits were very high (93% for the HA kit, 100% for the NP kit). The test sensitivities for detection of H1N1 pdm were 85.5% with the anti-NP antibody, 49.4% with the anti-HA antibody, and 79.5% with a commercially available influenza A virus detection assay. Use of the anti-NP antibody could allow the rapid and accurate diagnosis of H1N1 pdm infections.

Inactivation of parvovirus B19 by liquid heating incorporated in the manufacturing process of human intravenous immunoglobulin preparations

Several reports have suggested the possible transmission of human parvovirus B19 (B19) through the administration of plasma derivatives that had undergone virus inactivation by various types of heat treatment. However, none of the reports evaluated and discussed the inactivation of B19 by the heat treatment that is implemented in the individual manufacturing processes of such products. The present study evaluated the ability to inactivate B19 of liquid‐heat treatment at 60°C for 10 h that was incorporated in the manufacturing process of intravenous human immunoglobulin preparations. The results showed that B19 was rapidly inactivated under the conditions used for the liquid‐heat treatment.

Significant neutralizing activity of human immunoglobulin preparations against pandemic 2009 H1N1

The influenza-like illness that began in the Unites States and Mexico was first reported by the World Health Organization (WHO) on 24 April, 2009, and declared a phase 6 pandemic on 11 June. As of 6 July 2009, over 90 000 cases and more than 400 deaths in some 120 countries had been confirmed (WHO, 2009). Importantly, on July 8th, the WHO announced that oseltamivir (Tamiflu)-resistant viruses had been identified in Denmark, Japan and Hong Kong (WHO, 2009). The pandemic virus 2009 H1N1 was a triple reassortant of human-, swine- and avian-derived influenza A virus segments and the HA gene was classified as being of swine-origin (Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, (2009). Evidence is accumulating that specific IgG antibodies against this virus are present in certain populations, especially the elderly (Itoh et al, 2009). However, Katz et al (2009) reported that cross reactive IgG against a pandemic influenza virus (A/California/04/2009) was found in no serum specimens of children aged 6 months–9 years old, 8% of samples from 5- to 9-year olds, 9% of samples from 18- to 64-year olds, 6% of samples of 18- to 40-year olds and 33% of samples of those over 60 years old, suggesting that immunoglobulin preparations derived from pooled plasma from over 10 000 healthy donors could contain such cross reactive IgG. The present study evaluated haemagglutinin-inhibition (HI) and virus neutralization (VN) activities against 2009 H1N1 and seasonal H1N1 as a positive control in intravenous human immunoglobulin (IVIG) preparations manufactured in 1999 and 2008. An influenza A/H1N1 vaccine strain (A/New Caledonia/20/99), a clinical isolate of A/H1N1 (A/Osaka/16/2008), a classical swine isolate of A/H1N1 (A/Swine/Hokkaido/2/1981) and a pandemic influenza isolate of A/H1N1 (A/Osaka/168/2009 H1N1 pdm) were used in this study. Three lots (Lot. A, B and C) of IVIG derived from pooled plasma collected in Japan and manufactured in 2008 (IVIG2008JP, ‘Kenketsu Venoglobulin®-IH Yoshitomi; Benesis Corp., Osaka, Japan’) were also used. In addition, two lots of IVIG that were manufactured in 1999, derived from plasma pooled collected in Japan and the USA (IVIG1999JP ‘Kenketsu Venoglobulin®-IH’, IVIG1999US ‘Venoglobulin®-IH; Yoshitomi Pharmaceutical Industries, Ltd. at the time, currently Benesis Corp.’), were used. The viruses were propagated in Madin-Darby canine kidney (MDCK) cells or in the allantoic cavity of chicken embryonated eggs. The culture media and the allantoic fluids were stored at −80°C prior to use. Infectivity, as infectious focus-forming units (FFU) per ml, was titrated in MDCK cells using peroxidase and an anti-peroxidase (PAP) staining technique (Okuno et al, 1990). The haemagglutinin-inhibition (HI) test using 0·75% guinea pig red blood cells was carried out as described previously (Okuno et al, 1993). The results were expressed as the reciprocal of the highest dilution of the culture medium to show inhibition. The virus neutralization (VN) test was carried out as described (Okuno et al, 1990). Briefly, IVIG was diluted twofold with serum-free medium. The diluted IVIG (50 μl) was mixed with 100 FFU (50 μl) of virus, then applied to MDCK cells in a 96-well microplate. After culturing, the cells were fixed with ethanol and stained by PAP as above. The results were expressed as the reciprocal of the dilution giving 50% neutralization. Intravenous human immunoglobulins were manufactured using plasma pooled from over 10 000 healthy donors. The HI and VN activities of IVIGs were titrated against pandemic, seasonal human and swine influenza A viruses (Table I). Of note, both the 1999 and 2008 IVIGs were shown to have anti pandemic and classical swine influenza A/H1N1 virus titres with HI (×4–×8) and VN (×32–×64). The 2008 IVIGs showed titres against the vaccine strain A/New Caledonia/20/99, which was isolated in 1999, with HI (×160–×320) and VN (×640–×1280), while the 1999 IVIGs showed titres with HI (×10–×40) and VN (×32–×128). These results suggested that the IVIG derived from the pooled plasma contained a certain amount of functional IgG, including IgG against pandemic or classical swine influenza A/H1N1. Of note, such IgG titres were slightly higher in the IVIG2008JP products compared with IVIG1999JP. However, the titres were slightly higher in IVIG1999US than in IVIG1999JP. Higher titres against the vaccine and clinical strains were observed in IVIG1999US than IVIG1999JP. Interestingly, the difference in the increase in titres against the vaccine strain was much greater between the products manufactured in 2008 and 1999 than between the others. This difference seems to be an outcome of vaccination. Our preliminary results showed a HI titre >×40 in 1·2% (7/580), ×20 in 3·1% (18/580) and ×10 in 4·3% (25/580), indicating the possible production of hyperimmune globulin with these sources of plasma collected in 2008, Japan. Table I Cross reactivity of several lots of IVIG against pandemic 2009, classical swine and seasonal H1N1 viruses.

Infectious prion protein in the filtrate even after 15nm filtration

The evaluation of the removal efficacy during manufacturing is important for the risk assessment of plasma products with respect to possible contamination by infectious prions, as recently reported in several papers on the potential for prion transmission through plasma products. Here, we evaluated a virus removal filter which has 15 nm pores. An antithrombin sample immediately prior to nano-filtration was spiked with prion material prepared in two different ways. The removal (log reduction factor) of prion infectivity using animal bioassays was >or=4.72 and 4.00 in two independent filtrations. However, infectivity was detected in both the pellet and supernatant following ultracentrifugation of the 15 nm filtered samples, indicating difficulty in complete removal. The data supports the conclusion that a certain amount of infectious prion protein is present as a smaller and/or soluble form (less than approximately 15 nm in diameter).