Satoshi Hiroi

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.

Isolation and characterization of a novel recombinant human adenovirus species D.

A novel recombinant human adenovirus (HAdV) species D was isolated from the stool of a pharyngitis patient in Japan and genetic characterization was performed by sequencing variable regions between HAdV types. The nucleotide sequences of the penton base gene and loops 1 and 2 in the hexon gene showed 100% identity with that of the recently identified HAdV-56. Although we observed greatest identity for the entire hexon gene sequence with that of HAdV-56, we noted even greater similarity between the partial nucleotide sequence of the conserved region 4 and that of HAdV-37. Furthermore, the fibre gene and early region 3 sequences were completely identical to that of HAdV-37. These results suggest that the strain is a novel adenovirus related to HAdV-37 and HAdV-56.

Detection of respiratory viruses in gargle specimens of healthy children

Abstract Background Respiratory tract viral infection is one of the most common and important diseases in children. Polymerase chain reaction (PCR) tests are often used to detect viruses in samples, it is difficult to interpret the clinical significance of PCR positivity, which may reflect a past, imminent or active asymptomatic infection due to their high sensitivity. Although single respiratory viruses have been detected in samples from children with symptoms, other respiratory viruses can also be detected simultaneously. However, the clinical importance of these findings for the symptoms is not known. Objectives To investigate the prevalence of respiratory viruses among children without any symptoms such as acute respiratory illness and/or fever. Study design From week twenty-five 2013 to week twenty-six 2014, gargle samples were collected from children once a week and these samples were subjected to real-time PCR to detect respiratory viruses. On each sampling day, we asked the parents about their children’s health condition. Results Among the 286 samples collected, 200 were from asymptomatic children. In the asymptomatic condition, human parechovirus, adenovirus, enterovirus, rhinovirus, coronavirus 229E and HKU1 were observed in 45 episodes. In samples from symptomatic children, parainfluenza viruses, respiratory syncytial virus and coronavirus OC43 were detected in addition to those mentioned above. Conclusions Various viruses of different species were detected in the specimens from the children regardless of their health status. It might be speculated that host factors such as the function of the immune system influence the clinical outcome of the infection. However, this needs to be studied further.

Seasonal variations of respiratory viruses and etiology of human rhinovirus infection in children

Abstract Background Using the polymerase chain reaction (PCR) method it is possible to detect uncultivable viruses and discover multiple viral infections. However, the clinical importance of these findings in relation to symptoms is not known. Objectives The seasonal fluctuations of respiratory viruses and the clinical outcomes of single infections and dual infections were investigated. Study design Nasal aspirate samples were obtained from outpatients and inpatients of a children’s hospital and these samples were subjected to real-time PCR to detect 16 respiratory viruses. Seasonal variations of the 16 viruses and the clinical outcomes such as wheezing, the need for oxygenation and prolonged hospitalization of patients with single viral infections and multiple infections were determined for the 5 most often detected viruses. Results Among 512 specimens analyzed, one or more viruses were detected in 424 (83%) specimens. Two or more viruses were detected in 160 samples (31% of all samples). The epidemic peaks of the viruses did not coincide with each other. Rhinoviruses were the most frequently detected viruses and their coinfection rates were also higher. However, the disease severity in the lower respiratory tract did not differ in most respiratory viral infections regardless of whether there was single infection or dual infection with a rhinovirus and other respiratory virus. Conclusions Seasonal distribution was seen for each virus. There were no significant differences in clinical symptoms in the children studied. Because the infection of rhinoviruses is the common occurrence in children, it is hypothesized that the factors related to disease severity are mainly the underlying conditions of the children.

Trivalent influenza vaccine-induced antibody response to circulating influenza a (H3N2) viruses in 2010/11 and 2011/12 seasons

To evaluate antibody response induced by trivalent inactivated influenza vaccine (TIV) against circulating influenza A (H3N2) strains in healthy adults during the 2010/11 and 2011/12 seasons, a hemagglutination-inhibition (HI) assay was utilized to calculate geometric mean antibody titer (GMT), seroprotection rate (post vaccination HI titers of ≥1 :40), and seroresponse rate (4-fold increase in antibody level). In the 2010/11 season, GMT increased 1.8- to 2.0-fold following the first dose of TIV against 3 circulating strains and 2.2-fold following the second compared to before vaccination. The seroresponse rate ranged from 22% to 26% following the first dose of TIV and from 31% to 33% following the second (n = 54 ). The seroprotection rate increased from a range of 6% to 13% to a range of 26% to 33% following the first dose of TIV and to a range of 37% to 42% following the second (n = 54 ). In the 2011/12 season, GMT increased 1.4-fold against A/Osaka/110/2011 and 1.8-fold against A/Osaka/5/2012. For A/Osaka/110/2011, the seroresponse rate was 29%, and the seroprotection rate increased from 26% to 55% following vaccination (n = 31 ). For A/Osaka/5/2012, the seroresponse rate was 26%, and the seroprotection rate increased from 68% to 84% following vaccination (n = 31 ). HI assays with reference antisera demonstrated that the strains in the 2011/12 season were antigenically distinct from vaccine strain (A/Victoria/210/2009). In conclusion, the vaccination increased the seroprotection rate against circulating H3N2 strains in the 2010/11 and 2011/12 seasons. Vaccination of TIV might have potential to induce reactive antibodies against antigenically distinct circulating H3N2 viruses.

Human monoclonal antibodies neutralizing influenza virus A/H1N1pdm09 and seasonal A/H1N1 strains - Distinct Ig gene repertoires with a similar action mechanism.

Influenza virus causes acute respiratory infection in humans, and is a major public health concern globally. Antibodies play a central role in host protection against influenza virus. We isolated human monoclonal antibodies (hMAb) 206-2-4 and 201-6-8 by a human hybridoma protocol that neutralized various but distinct influenza virus (IFV) A/H1N1 strains, including 2009 pandemic strains. The half-inhibitory concentration of 206-2-4 and 201-6-8 against A/H1N1pdm09 strains was 2-100ng/mL and 5-20μg/mL, respectively. Prophylactic and therapeutic potencies of 206-2-4 were demonstrated in a mouse model of IFV infection at i.p. dosages of 0.25 and 2.5mg/kg, respectively, suggesting that 206-2-4 is one of the most potent hnMAbs against IFV reported thus far. The Ig genes of 206-2-4 and 201-6-8 were originated from distinct germ line repertoires, and accompanied by 63 and 23 somatic hypermutations, respectively. The hemagglutination inhibitory activity indicated that the mechanism of neutralization was to interfere the virus-receptor interaction. The binding epitope of the two antibodies was mapped to hemagglutinin 1 (HA1) amino acid residues 111-120. Additional interaction between the antibody and the HA1 globular head was necessary for neutralization. Such hnMAbs bearing distinct binding epitope have been rarely reported. The potency is likely due to the coverage of a wide surface area of HA protein by these hnMABs. IFV is a highly variable. Our knowledge on the mechanisms by which these cross-reactive hnMAbs function should help design a novel immunogen for the development of a vaccine effective against broader spectrum of IFV strains.

Surveillance of adenovirus respiratory infections in children in Osaka, Japan

Human adenovirus (HAdV) strains isolated from respiratory specimens of 139 children were analyzed to evaluate the endemic situation of HAdV infections in Osaka, Japan, between 2008 and 2015. The cases increased during spring and winter, and the infections were confirmed mainly in children aged ≤ 5 years, comprising 91.9% of the total population examined. Molecular typing of the isolates revealed that the most common types belonged to HAdV-B and -C. Co-infection of HAdV-C1 and -C2 was also confirmed in a case. The median age of HAdV-E cases was higher than that of the HAdV-B and -C cases. These results revealed age and seasonal distribution of respiratory HAdV infections in children from Osaka, and indicate that majority of these children might have acquired immunity through endemic HAdV infection before reaching school age.