No association between inactivated influenza vaccination and influenza viral load at diagnosis among young Japanese children: An observational study of the 2013/2014 through 2017/2018 influenza seasons.

BACKGROUND: The association between inactivated influenza vaccination and viral load in young children remains unclear. METHODS: During the 2013/2014 to 2017/2018 influenza seasons in Japan, children under 6 years of age with pre-defined influenza-like illness and influenza-positive status by real-time RT-PCR were recruited at pediatric clinics for this observational study. Influenza viral load was measured for the most predominant subtype/lineage in each season. Using median dichotomized viral load as an outcome, a multilevel logistic regression model was applied to estimate the multivariable adjusted odds ratio (MOR) and 95% confidence interval (CI) for higher viral load. RESULTS: A total of 1,185 influenza-positive children were analyzed. The median log10 viral load copy number (copies per milliliter) was 5.5 (interquartile range, 4.6 to 6.1) and did not differ by vaccination status: 5.5 for unvaccinated, 5.7 for one dose, and 5.5 for two doses (p = 0.67). The MOR of vaccinated (one or two doses) versus unvaccinated children was 1.19 (95% CI: 0.86-1.64). Other factors showing significant associations with higher viral load were positive results for A(H1N1)pdm09 and A(H3N2) in comparison with B/Yamagata. The respective MORs were 3.25 (95% CI: 2.28-4.64) and 1.81 (95% CI: 1.32-2.49). Significantly elevated MORs against higher viral load were also observed for higher body temperature at influenza diagnosis and shorter duration from fever onset to specimen collection. CONCLUSION: No association was observed between inactivated-influenza vaccination and viral load at influenza-positive diagnosis. Influenza subtype/lineage, body temperature, and time elapsed since fever onset were significantly associated with viral load.

Vaccine-induced neutralizing antibodies against SARS-CoV-2 Omicron variant isolated in Osaka, Japan.

To study vaccine-induced neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants isolated in Osaka, Japan, microneutralization tests were performed on serum samples from 32subjects who received a second dose of vaccination, and 10 of those who received the third dose of vaccination. Geometric mean titres (GMTs) for the D614G strain, Alpha variant, Delta variant, and Omicron BA.1 of the subjects after the second dose of vaccination were 19.5, 21.8, 6.3 and 2.0, respectively. The GMT for the Delta variant was significantly lower than that for the D614G strain and Alpha variant, and the GMT for the Omicron BA.1 was significantly lower than that for the Delta variant. Among the subjects who received three doses of vaccination, the GMTs for the Omicron BA.1 (62.8) and BA.2 (38.6) were significantly higher than that for the Omicron BA.1 after the second dose. Thus, in the present study, the second dose of vaccination induced neutralizing antibodies against SARS-CoV-2 strains, and the reactivity of neutralizing antibodies to the variants was thought to be enhanced by the third dose of vaccination. The serum samples used in this study will be useful in evaluating the reactivity of vaccine-induced antibodies to newly emerging variants.

Influence of Prior Influenza Vaccination on Current Influenza Vaccine Effectiveness in Children Aged 1 to 5 Years.

BACKGROUND: Although annual influenza vaccination is an important strategy used to prevent influenza-related morbidity and mortality, some studies have reported the negative influence of prior vaccination on vaccine effectiveness (VE) for current seasons. Currently, the influence of prior vaccination is not conclusive, especially in children. METHODS: We evaluated the association between current-season VE and prior season vaccination using a test-negative design in children aged 1-5 years presenting at nine outpatient clinics in Japan during the 2016/17 and 2017/18 influenza seasons. Children with influenza-like illness were enrolled prospectively and tested for influenza using real-time RT-PCR. Their recent vaccination history was categorized into six groups according to current vaccination doses (0/1/2) and prior vaccination status (unvaccinated = 0 doses/vaccinated = 1 dose or 2 doses): (1) 0 doses in the current season and unvaccinated in prior seasons (reference group); (2) 0 doses in the current season and vaccinated in a prior season; (3) 1 dose in the current season and unvaccinated in a prior season; (4) 1 dose in the current season and vaccinated in a prior season; (5) 2 doses in the current season and unvaccinated in a prior season, and (6) 2 doses in the current season and vaccinated in a prior season. RESULTS: A total of 799 cases and 1196 controls were analyzed. The median age of the subjects was 3 years, and the proportion of males was 54%. Overall, the vaccination rates (any vaccination in the current season) in the cases and controls were 36% and 53%, respectively. The VEs of the groups were: (2) 29% (95% confidence interval: -25% to 59%); (3) 53% (6% to 76%); (4) 70% (45% to 83%); (5) 56% (32% to 72%), and (6) 61% (42% to 73%). The one- and two-dose VEs of the current season were significant regardless of prior vaccination status. The results did not differ when stratified by influenza subtype/lineage. CONCLUSION: Prior vaccination did not attenuate the current-season VE in children aged 1 to 5 years, supporting the annual vaccination strategy.

Infectivity assay for detection of SARS-CoV-2 in samples from patients with COVID-19.

Since the coronavirus disease 2019 (COVID-19) outbreak, laboratory diagnosis has mainly been conducted using reverse-transcription polymerase chain reaction (RT-PCR). Detecting the presence of an infectious virus in the collected sample is essential to analyze if a person can transmit infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there have been no quantitative investigations conducted for infectious SARS-CoV-2 in clinical samples. Therefore, in the present study, a rapid and simple focus-forming assay using the peroxidase-antiperoxidase technique was developed to quantify infectious SARS-CoV-2 titers in 119 samples (n = 52, nasopharyngeal swabs [NPS]; n = 67, saliva) from patients with COVID-19. Furthermore, the study findings were compared with the cycle threshold (Ct) values of real-time RT-PCR. The infectious virus titers in NPS samples and Ct values were inversely correlated, and no infectious virus could be detected when the Ct value exceeded 30. In contrast, a low correlation was observed between the infectious virus titers in saliva and Ct values (r = -0.261, p = 0.027). Furthermore, the infectious virus titers in the saliva were significantly lower than those in the NPS samples. Ten days after the onset of COVID-19 symptoms, the infectious virus was undetectable, and Ct values were more than 30 in NSP and saliva samples. The results indicate that patients whose symptoms subsided 10 days after onset, with Ct values more than 30 in NSP and saliva samples, were less likely to infect others.

Comparison of immunogenicity between candidate influenza A(H3N2) virus vaccine strains in Japan: A randomized controlled trial using a monovalent vaccine of A/Saitama/103/2014 (CEXP-002) and A/Hong Kong/4801/2014 (X-263).

BACKGROUND: For the 2017-18 influenza season, A/Saitama/103/2014 (CEXP-002) (Saitama strain) was antigenically more similar to prior circulating strains than A/Hong Kong/4801/2014 (X-263) (Hong Kong strain) in a ferret model and was selected as the A(H3N2) vaccine virus strain in Japan. However, the Saitama strain grew poorly, and the Japanese government switched to the Hong Kong strain, raising public concerns of poor effectiveness. To enhance understanding of the correlation between antigenicity in experimental models and immunogenicity, as a surrogate measure of vaccine effectiveness, in the human population, we compared the immunogenicity of specially-prepared single dose monovalent influenza A(H3N2) vaccines containing the Saitama or the Hong Kong strain. METHODS: A randomized controlled trial of 100 healthy adults aged 20-64 years (n = 50/group) was conducted. Virus neutralization assay was performed on sera from days 0 (pre-vaccination) and 21 (post-vaccination). Geometric mean titer (GMT), mean fold rise (MFR), seroconversion proportion (SCP), and seroprotection proportion (SPP) were calculated for vaccine strains and a representative circulating A(H3N2) virus strain (A/Osaka/188/2017). RESULTS: For the Hong Kong strain, post-vaccination GMT was significantly higher in the Hong Kong vaccine recipients (1:546 vs 1:260, p < 0.01), but MFR, SCP, and SPP were similar for both vaccine groups. For the Saitama strain, post-vaccination GMT (1:116 vs 1:61, p = 0.01) and SPP (86% vs 68%, p = 0.03) were significantly higher in the Hong Kong vaccine recipients, but MFR and SCP were similar for both vaccine groups. Against A/Osaka/188/2017, post-vaccination GMT and MFR were similar in both vaccine groups, but SCP (32% vs 4%, p < 0.01) and SPP (28% vs. 6%, p < 0.01) were significantly higher in the Hong Kong vaccine recipients. CONCLUSION: The Hong Kong vaccine induced better or equivalent immunogenicity in comparison to the Saitama vaccine. Our trial showed that antigenic similarity in experimental models does not necessarily correlate with immunogenicity in the human population. CLINICAL TRIAL REGISTRATION: UMIN000029293.

Comparison of the Pathogenicity in Mice of A(H1N1)pdm09 Viruses Isolated between 2009 and 2015 in Japan.

The A(H1N1)pdm09 virus emerged in 2009 and continues to circulate in human populations. Recent A(H1N1)pdm09 viruses, that is, A(H1N1)pdm09 viruses circulating in the post-pandemic era, can cause more or less severe infections than those caused by the initial pandemic viruses. To evaluate the changes in pathogenicity of the A(H1N1)pdm09 viruses during their continued circulation in humans, we compared the nucleotide and amino acid sequences of ten A(H1N1)pdm09 viruses isolated in Japan between 2009 and 2015, and experimentally infected mice with each virus. The severity of infection caused by these Japanese isolates ranged from milder to more severe than that caused by the prototypic pandemic strain A/California/04/2009 (CA04/09); however, specific mutations responsible for their pathogenicity have not yet been identified.

Surveillance of Adenovirus Respiratory Infections in Children from 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.

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

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.

Detection of respiratory viruses in gargle specimens of healthy children.

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.

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.

Molecular epidemiology of human adenoviruses d associated with epidemic keratoconjunctivitis in Osaka, Japan, 2001-2010.

Isolation of novel types of human adenovirus D (HAdV-53, -54, and -56) from keratoconjunctivitis patients has been reported since 2008. We examined the molecular epidemiology of HAdV-D strains using 39 field isolates collected from epidemic keratoconjunctivitis (EKC) patients from 2001 to 2010 in the province of Osaka, Japan. The molecular types were analyzed by sequencing partial penton base gene, loop 1 in the hexon, and complete fiber genes. Of the 39 isolates, the majority were HAdV-19 (14/39, 35.9%) and -37 (13/39, 33.3%), followed by -53 (4/39, 10.3%) and -54 (8/39, 20.5%). Analysis of annual distribution showed that HAdV-19 and -37 were detected before 2004, whereas HAdV-53 and -54 were first identified in 2001 and 2003, respectively, and persistently detected during the study period. It is noted that both HAdV-53 and -54 isolates were misclassified by the serological method. Altogether, the molecular analysis elucidated the epidemiology of HAdV-D and presence of novel types from the early 2000s in Osaka. Further genetic analysis of serologically classified HAdV-D isolates may provide insights into the epidemiology of EKC.

[Characterization of a nonspecific inhibitor found in human sera raised against the 2006/07 influenza vaccine strain A/Hiroshima/52/2005 (H3N2) virus].

The serology of influenza viruses typically uses hemagglutination inhibition (HI) or the neutralization test (NT). However, the sera of many humans and animals contain nonspecific inhibitors of hemagglutinin that must be inactivated or removed from the serum before use in the HI assay. Any nonspecific inhibitor in human serum is typically inactivated by pre-treatment with receptor-destroying enzyme (RDE). However, during the 2006/07 influenza circulating season, we observed that influenza vaccine strain A/Hiroshima/52/ 2005 (H3N2) exhibited susceptibility to an RDE-resistant inhibitor in human serum. We report herein on a preliminary characterization of this inhibitor, including the development of a novel inhibitor-inactivating technique for pre-treatment of human serum to be used for HI with the A/Hiroshima/52/2005 (H3N2) virus.

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.

Development of an immunochromatographic assay specifically detecting pandemic H1N1 (2009) influenza virus.

The pandemic caused by a new type of influenza virus, pandemic H1N1 (2009) influenza virus A (AH1pdm), has had a major worldwide impact. Since hemagglutinin (HA) genes are among the most specific genes in the influenza virus genome, AH1pdm can be definitively diagnosed by viral gene analysis targeting the HA genes. This type of analysis, however, cannot be easily performed in clinical settings. While commercially available rapid diagnosis kits (RDKs) based on immunochromatography can be used to detect nucleoproteins (NPs) of influenza A and B viruses in clinical samples, there are no such kits that are specific for AH1pdm. We show here that an RDK using a combination of monoclonal antibodies against NP can be used to specifically detect AH1pdm. The RDK recognized AH1pdm virus isolates but did not recognize seasonal H1N1 and H3N2 and influenza B viruses, indicating that the specificity of the RDK is 100%. A parallel comparison of RDK with a commercial influenza A/B virus kit revealed that both types of kits had equal sensitivities in detecting their respective viruses. Preliminary evaluation of clinical samples from 5 individuals with PCR-confirmed human AH1pdm infection showed that the RDK was positive for all samples, with the same detection intensity as that of a commercial influenza A/B virus kit. This RDK, together with a new vaccine and the stockpiling of anti-influenza drugs, will make aggressive measures to contain AH1pdm infections possible.

Combined administration of (L)-cystine and (L)-theanine enhances immune functions and protects against influenza virus infection in aged mice.

Cell-mediated and humoral immune responses are attenuated with aging. Intracellular glutathione (GSH) levels also decrease with aging. Previously, we have reported that combined administration of (L)-cystine and (L)-theanine enhances antigen-specific IgG production, partly through augmentation of GSH levels and T helper 2-mediated responses in 12-week-old mice. These findings suggest that combined administration of (L)-cystine and (L)-theanine to aged mice improves immune responses via increase of GSH synthesis. Here, we examined the effects of combined administration of (L)-cystine and (L)-theanine on antigen-specific antibody production and influenza virus infection in aged mice. Combined administration of these amino acids for 14 days before primary immunization significantly enhanced the serum antigen-specific IgM and IgG levels in 24-month-old mice. Furthermore, 13-month-old mice co-treated with these amino acids orally for 10 days had significantly lower lung viral titers than controls at 6 days after influenza virus infection. In addition, this co-treatment also significantly prevented the weight loss associated with infection. Enhancement of anti-influenza-virus IgG antibodies by combined administration of (L)-cystine and (L)-theanine was seen 10 days after infection. The significantly elevated serum interleukin-10/interferon-gamma ratio and gamma-glutamylcysteine synthetase mRNA expression, which is the rate-limiting enzyme of GSH synthesis, in the spleen 3 days after infection may have contributed to the observed beneficial effects. These results suggest that combined administration of (L)-cystine and (L)-theanine enhances immune function and GSH synthesis which are compromised with advanced age, and may become a useful strategy in healthy aging.