Biphasic Waning of Hemagglutination Inhibition Antibody Titers After Influenza Vaccination in Children.

We explored the potential for a biphasic pattern in waning of antibody titers after influenza vaccination. We collected blood samples in a randomized controlled trial of influenza vaccination in children and tested them with hemagglutination inhibition assays for influenza A(H3N2) and influenza B/Victoria lineage. Using piecewise log-linear mixed-effects models, we found evidence for a faster initial waning of antibody titers for the first 1-2 years after vaccination and then slower longer-term declines. Children with higher postvaccination titers had faster antibody decay.

Relative incidence and individual-level severity of seasonal influenza A H3N2 compared with 2009 pandemic H1N1.

BACKGROUND: Two subtypes of influenza A currently circulate in humans: seasonal H3N2 (sH3N2, emerged in 1968) and pandemic H1N1 (pH1N1, emerged in 2009). While the epidemiological characteristics of the initial wave of pH1N1 have been studied in detail, less is known about its infection dynamics during subsequent waves or its severity relative to sH3N2. Even prior to 2009, few data was available to estimate the risk of severe outcomes following infection with one circulating influenza strain relative to another. METHODS: We analyzed antibodies in quadruples of sera from individuals in Hong Kong collected between July 2009 and December 2011, a period that included three distinct influenza virus epidemics. We estimated infection incidence using these assay data and then estimated rates of severe outcomes per infection using population-wide clinical data. RESULTS: Cumulative incidence of infection was high among children in the first epidemic of pH1N1. There was a change towards the older age group in the age distribution of infections for pH1N1 from the first to the second epidemic, with the age distribution of the second epidemic of pH1N1 more similar to that of sH3N2. We found no serological evidence that individuals were infected in both waves of pH1N1. The risks of excess mortality conditional on infection were higher for sH3N2 than for pH1N1, with age-standardized risk ratios of 2.6 [95% CI: 1.8, 3.7] for all causes and 1.5 [95% CI: 1.0, 2.1] for respiratory causes throughout the study period. CONCLUSIONS: Overall increase in clinical incidence of pH1N1 and higher rates of severity in older adults in post pandemic waves were in line with an age-shift in infection towards the older age groups. The absence of repeated infection is good evidence that waning immunity did not cause the second wave. Despite circulating in humans since 1968, sH3N2 is substantially more severe per infection than the pH1N1 strain. Infection-based estimates of individual-level severity have a role in assessing emerging strains; updating seasonal vaccine components; and optimizing of vaccination programs.

Glycomic analysis of human respiratory tract tissues and correlation with influenza virus infection.

The first step in influenza infection of the human respiratory tract is binding of the virus to sialic (Sia) acid terminated receptors. The binding of different strains of virus for the receptor is determined by the α linkage of the sialic acid to galactose and the adjacent glycan structure. In this study the N- and O-glycan composition of the human lung, bronchus and nasopharynx was characterized by mass spectrometry. Analysis showed that there was a wide spectrum of both Sia α2-3 and α2-6 glycans in the lung and bronchus. This glycan structural data was then utilized in combination with binding data from 4 of the published glycan arrays to assess whether these current glycan arrays were able to predict replication of human, avian and swine viruses in human ex vivo respiratory tract tissues. The most comprehensive array from the Consortium for Functional Glycomics contained the greatest diversity of sialylated glycans, but was not predictive of productive replication in the bronchus and lung. Our findings indicate that more comprehensive but focused arrays need to be developed to investigate influenza virus binding in an assessment of newly emerging influenza viruses.

Phenotypic and functional characterization of human γδ T-cell subsets in response to influenza A viruses.

Like αß T cells, human γδ T cells also have different subsets with distinct characteristics. Whether human Vγ9Vδ2 T cells have functionally different subsets in response to influenza A (fluA) viruses remains unknown. In this study, we show for the first time that both central (CD45RA(-)CD27(+)) and effector (CD45RA(-)CD27(-)) memory Vγ9Vδ2 T cells have similar levels of immediate interferon (IFN) γ and cytotoxic responses to human and avian fluA virus-infected cells. In contrast, CD56(+) Vγ9Vδ2 T cells have significantly higher cytotoxicity against fluA virus-infected cells compared with their CD56(-) counterparts, whereas both subsets have similar IFN-γ responses. We further demonstrate that the CD16-dependent degranulation pathway, but not antibody-dependent cell-mediated cytotoxicity, contribute to the superior cytotoxicity of CD56(+) Vγ9Vδ2 T cells. Our study provides further evidence for the phenotypic and functional characterization of human Vγ9Vδ2 T-cell subsets during fluA virus infection and may help improve the γδ T-cell-based immunotherapy for viral infection.

Impact of host genetic polymorphisms on response to inactivated influenza vaccine in children.

In randomized controlled trials of influenza vaccination, 550 children received trivalent-inactivated influenza vaccine, permitting us to explore relationship between vaccine response and host single nucleotide polymorphisms (SNPs) in 23 candidate genes with adjustment of multiple testing. For host SNPs in TLR7-1817G/T (rs5741880), genotype GT was associated with lower odds (OR: 0.22, 95% CI: 0.09, 0.53) of have post-vaccination hemagglutination-inhibiting (HAI) titers ≥40, compared with genotype GG and TT combined under the over-dominant model. For host SNPs in TLR8-129G/C (rs3764879), genotype GT was associated with lower odds (OR: 0.47; 95% CI: 0.28, 0.80) of have post vaccination HAI titers ≥40 compared with genotype GG and AA combined under the over-dominant model. Our results could contribute to the development of better vaccines that may offer improved protection to all recipients.

Reconstructing antibody dynamics to estimate the risk of influenza virus infection.

For >70 years, a 4-fold or greater rise in antibody titer has been used to confirm influenza virus infections in paired sera, despite recognition that this heuristic can lack sensitivity. Here we analyze with a novel Bayesian model a large cohort of 2353 individuals followed for up to 5 years in Hong Kong to characterize influenza antibody dynamics and develop an algorithm to improve the identification of influenza virus infections. After infection, we estimate that hemagglutination-inhibiting (HAI) titers were boosted by 16-fold on average and subsequently decrease by 14% per year. In six epidemics, the infection risks for adults were 3%-19% while the infection risks for children were 1.6-4.4 times higher than that of younger adults. Every two-fold increase in pre-epidemic HAI titer was associated with 19%-58% protection against infection. Our inferential framework clarifies the contributions of age and pre-epidemic HAI titers to characterize individual infection risk.

Effect modification of environmental factors on influenza-associated mortality: a time-series study in two Chinese cities.

BACKGROUND: Environmental factors have been associated with transmission and survival of influenza viruses but no studies have ever explored the role of environmental factors on severity of influenza infection. METHODS: We applied a Poisson regression model to the mortality data of two Chinese metropolitan cities located within the subtropical zone, to calculate the influenza associated excess mortality risks during the periods with different levels of temperature and humidity. RESULTS: The results showed that high absolute humidity (measured by vapor pressure) was significantly (p < 0.05) associated with increased risks of all-cause and cardiorespiratory deaths, but not with increased risks of pneumonia and influenza deaths. The association between absolute humidity and mortality risks was found consistent among the two cities. An increasing pattern of influenza associated mortality risks was also found across the strata of low to high relative humidity, but the results were less consistent for temperature. CONCLUSIONS: These findings highlight the need for people with chronic cardiovascular and respiratory diseases to take extra caution against influenza during hot and humid days in the subtropics and tropics.

Effects of oseltamivir treatment on duration of clinical illness and viral shedding and household transmission of influenza virus.

BACKGROUND: Large clinical trials have demonstrated the therapeutic efficacy of oseltamivir against influenza. We assessed the indirect effectiveness of oseltamivir in reducing secondary household transmission in an incident cohort of influenza index patients and their household members. METHODS: We recruited index outpatients whose rapid test results were positive for influenza from February through September 2007 and January through September 2008. Household contacts were followed up for 7-10 days during 3-4 home visits to monitor symptoms. Nose and throat swabs were collected and tested for influenza by reverse-transcription polymerase chain reaction or viral culture. RESULTS: We followed up 384 index patients and their household contacts. Index patients who took oseltamivir within 24 h of symptom onset halved the time to symptom alleviation (adjusted acceleration factor, 0.56; 95% confidence interval [CI], 0.42-0.76). Oseltamivir treatment was not associated with statistically significant reduction in the duration of viral shedding. Household contacts of index patients who had taken oseltamivir within 24 h of onset had a nonstatistically significant lower risk of developing laboratory-confirmed infection (adjusted odds ratio, 0.54; 95% CI, 0.11-2.57) and a marginally statistically significant lower risk of clinical illness (adjusted odds ratio, 0.52; 95% CI, 0.25-1.08) compared with contacts of index patients who did not take oseltamivir. CONCLUSIONS: Oseltamivir treatment is effective in reducing the duration of symptoms, but evidence of household reduction in transmission of influenza virus was inconclusive.

The infection attack rate and severity of 2009 pandemic H1N1 influenza in Hong Kong.

BACKGROUND: Serial cross-sectional data on antibody levels to the 2009 pandemic H1N1 influenza A virus from a population can be used to estimate the infection attack rates and immunity against future infection in the community. METHODS: From April through December 2009, we obtained 12,217 serum specimens from blood donors (aged 16-59 years), 2520 specimens from hospital outpatients (aged 5-59 years), and 917 specimens from subjects involved in a community pediatric cohort study (aged 5-14 years). We estimated infection attack rates by comparing the proportions of specimens with antibody titers ≥ 1:40 by viral microneutralization before and after the first wave of the pandemic. Estimates were validated using paired serum samples from 324 individuals that spanned the first wave. Combining these estimates with epidemiologic surveillance data, we calculated the proportion of infections that led to hospitalization, admission to the intensive care unit (ICU), and death. RESULTS: We found that 3.3% and 14% of persons aged 5-59 years had antibody titers ≥ 1:40 before and after the first wave, respectively. The overall attack rate was 10.7%, with age stratification as follows: 43.4% in persons aged 5-14 years, 15.8% in persons aged 15-19 years, 11.8% in persons aged 20-29 years, and 4%-4.6% in persons aged 30-59 years. Case-hospitalization rates were 0.47%-0.87% among persons aged 5-59 years. Case-ICU rates were 7.9 cases per 100,000 infections in persons aged 5-14 years and 75 cases per 100,000 infections in persons aged 50-59 years, respectively. Case-fatality rates were 0.4 cases per 100,000 infections in persons aged 5-14 years and 26.5 cases per 100,000 infections in persons aged 50-59 years, respectively. CONCLUSIONS: Almost half of all school-aged children in Hong Kong were infected during the first wave. Compared with school children aged 5-14 years, older adults aged 50-59 years had 9.5 and 66 times higher risks of ICU admission and death if infected, respectively.

Kinetics of viral load and antibody response in relation to COVID-19 severity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus 2019 (COVID-19) pneumonia. Little is known about the kinetics, tissue distribution, cross-reactivity, and neutralization antibody response in patients with COVID-19. Two groups of patients with RT-PCR-confirmed COVID-19 were enrolled in this study: 12 severely ill patients in intensive care units who needed mechanical ventilation and 11 mildly ill patients in isolation wards. Serial clinical samples were collected for laboratory detection. Results showed that most of the severely ill patients had viral shedding in a variety of tissues for 20-40 days after onset of disease (8/12, 66.7%), while the majority of mildly ill patients had viral shedding restricted to the respiratory tract and had no detectable virus RNA 10 days after onset (9/11, 81.8%). Mildly ill patients showed significantly lower IgM response compared with that of the severe group. IgG responses were detected in most patients in both the severe and mild groups at 9 days after onset, and remained at a high level throughout the study. Antibodies cross-reactive to SARS-CoV and SARS-CoV-2 were detected in patients with COVID-19 but not in patients with MERS. High levels of neutralizing antibodies were induced after about 10 days after onset in both severely and mildly ill patients which were higher in the severe group. SARS-CoV-2 pseudotype neutralization test and focus reduction neutralization test with authentic virus showed consistent results. Sera from patients with COVID-19 inhibited SARS-CoV-2 entry. Sera from convalescent patients with SARS or Middle East respiratory syndrome (MERS) did not. Anti-SARS-CoV-2 S and N IgG levels exhibited a moderate correlation with neutralization titers in patients' plasma. This study improves our understanding of immune response in humans after SARS-CoV-2 infection.

Estimation of the serial interval of influenza.

BACKGROUND: : Estimates of the clinical-onset serial interval of human influenza infection (time between onset of symptoms in an index case and a secondary case) are used to inform public health policy and to construct mathematical models of influenza transmission. We estimate the serial interval of laboratory-confirmed influenza transmission in households. METHODS: : Index cases were recruited after reporting to a primary healthcare center with symptoms. Members of their households were followed-up with repeated home visits. RESULTS: : Assuming a Weibull model and accounting for selection bias inherent in our field study design, we used symptom-onset times from 14 pairs of infector/infectee to estimate a mean serial interval of 3.6 days (95% confidence interval = 2.9-4.3 days), with standard deviation 1.6 days. CONCLUSION: : The household serial interval of influenza may be longer than previously estimated. Studies of the complete serial interval, based on transmission in all community contexts, are a priority.

Analysis of H5N1 avian influenza infections from wild bird surveillance in Hong Kong from January 2006 to October 2007.

Intensive surveillance of dead wild birds for H5N1 avian influenza infection is conducted in Hong Kong. Between January 2006 and October 2007 pooled cloacal and tracheal swabs from 17692 dead wild birds (from 16 different orders including 82 genera) were tested and 33 H5N1 highly pathogenic avian influenza viruses were isolated. No H5N1 infection has occurred in poultry farms since January 2003, or in live poultry markets in Hong Kong since November 2003 until a recent detection of H5N1 virus by surveillance of live poultry markets in June 2008. The gross and histopathology in the various H5N1-infected avian species is described, along with the performance of the virus isolation and polymerase chain reaction (PCR) tests used. This evaluation also included determination of virus titres and detection limits for the H5 haemagglutinin gene (H5)and matrix gene real-time reverse-transcription PCR tests in cloacal and tracheal swabs from 12 wild birds. The viruses isolated belonged to Clades 2.3.2 and 2.3.4, and within Clade 2.3.4 some clustering was evident based on H5 haemagglutinin gene haemagglutinating sequencing. There were no differences in the pathology findings between these subgroupings and the various diagnostic tests gave similar results for these viruses, except for a loss in sensitivity of the H5 real-time reverse-transcription PCR for several viruses in one cluster from birds submitted in February 2007. The use of multiple test methods was important in maintaining the diagnostic sensitivity for detecting avian influenza viruses with high genetic variability.

Avian influenza viruses in humans.

Past pandemics arose from low pathogenic avian influenza (LPAI) viruses. In more recent times, highly pathogenic avian influenza (HPAI) H5N1, LPAI H9N2 and both HPAI and LPAI H7 viruses have repeatedly caused zoonotic disease in humans. Such infections did not lead to sustained human-to-human transmission. Experimental infection of human volunteers and seroepidemiological studies suggest that avian influenza viruses of other subtypes may also infect humans. Viruses of the H7 subtype appear to have a predilection to cause conjunctivitis and influenza-like illness (ILI), although HPAI H7N7 virus has also caused fatal respiratory disease. Low pathogenic H9N2 viruses have caused mild ILI and its occurrence may be under-recognised for this reason. In contrast, contemporary HPAI H5N1 viruses are exceptional in their virulence for humans and differ from human seasonal influenza viruses in their pathogenesis. Patients have a primary viral pneumonia progressing to acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome. Over 380 human cases have been confirmed to date, with an overall case fatality of 63%. The zoonotic transmission of avian influenza is a rare occurrence, butthe greater public health concern is the adaptation of such viruses to efficient human transmission, which could lead to a pandemic. A better understanding of the ecology of avian influenza viruses and the biological determinants of transmissibility and pathogenicity in humans is important for pandemic preparedness.

Multiple sublineages of influenza A virus (H5N1), Vietnam, 2005-2007.

Phylogenetic analysis of influenza subtype H5N1 viruses isolated from Vietnam during 2005-2007 shows that multiple sublineages are present in Vietnam. Clade 2.3.4 viruses have replaced clade 1 viruses in northern Vietnam, and clade 1 viruses have been detected in southern Vietnam. Reassortment between these 2 sublineages has also occurred.

Heterologous influenza vRNA segments with identical non-coding sequences stimulate viral RNA replication in trans.

The initiation of transcription and replication of influenza A virus requires the 5' and 3' ends of vRNA. Here, the role of segment-specific non-coding sequences of influenza A virus on viral RNA synthesis was studied. Recombinant viruses, with the nonstructural protein (NS) segment-specific non-coding sequences replaced by the corresponding sequences of the neuraminidase (NA) segment, were characterized. The NS and NA vRNA levels in cells infected with these mutants were much higher than those of the wild type, whereas the NS and NA mRNA levels of the mutants were comparable to the wild-type levels. By contrast, the PB2 vRNA and mRNA levels of all the tested viruses were similar, indicating that vRNA with heterologous segment-specific non-coding sequences was not affected by the mutations. The observations suggested that, with the cooperation between the homologous 5' and 3'segment-specific sequences, the introduced mutations could specifically enhance the replication of NA and NS vRNA.

The effectiveness of influenza vaccination against medically-attended illnesses in Hong Kong across three years with different degrees of vaccine match, 2014-17.

BACKGROUND: Influenza vaccination is the most effective intervention to prevent influenza virus infections. Vaccine effectiveness (VE) can vary due to factors such as matching between vaccine strains and prevailing strains, age and other characteristics of the vaccine recipients. OBJECTIVE: To evaluate influenza VE against medically-attended illness in different age groups and against specific influenza types/subtypes in Hong Kong. METHODS: A test-negative study was conducted from December 2014 through August 2017 in 20 outpatient clinics. Patients at least 6 months of age presenting with at least two symptoms of acute respiratory illness, ARI (fever ≥37.8 °C, cough, sore throat, runny nose, headache, myalgia and phlegm) within 72 h of onset were tested for influenza virus by reverse transcription polymerase chain reaction (PCR). Vaccination history was assessed by self-report or medical records at the clinics. VE against medically-attended illness was estimated using conditional logistic regression for influenza PCR result versus vaccination history, matching by calendar time and adjusting for age, age-squared, sex, and chronic medical illness. Additional analyses examined VE by age group and by influenza type/subtype. RESULTS: We enrolled 2566 patients, of whom 1118 (43.6%) tested positive for influenza A or B virus by PCR. Test-positive subjects were generally older, more likely to present with one of the symptoms of ARI, and less likely to receive vaccination against influenza. VE estimates for influenza A(H1N1), A(H3N2), B/Yamagata and B/Victoria were 61.6% (95% confidence interval, CI: 21.8%, 81.1%), 26.4% (95% CI: -1.3%, 46.6%), 67.0% (95% CI: 25.9%, 85.3%), 60.4% (95% CI: 0.3%, 84.3%), respectively. Estimates of VE by age group were generally higher in adults aged 50-64 and lower among children and older adults. CONCLUSIONS: VE against medically-attended influenza was moderate in Hong Kong, confirming the impact of influenza vaccination in reducing disease burden. The reduced VE for influenza A(H3N2) is a continuing concern.

Whole transcriptome analysis reveals differential gene expression profile reflecting macrophage polarization in response to influenza A H5N1 virus infection.

BACKGROUND: Avian influenza A H5N1 virus can cause lethal disease in humans. The virus can trigger severe pneumonia and lead to acute respiratory distress syndrome. Data from clinical, in vitro and in vivo suggest that virus-induced cytokine dysregulation could be a contributory factor to the pathogenesis of human H5N1 disease. However, the precise mechanism of H5N1 infection eliciting the unique host response are still not well understood. METHODS: To obtain a better understanding of the molecular events at the earliest time points, we used RNA-Seq to quantify and compare the host mRNA and miRNA transcriptomes induced by the highly pathogenic influenza A H5N1 (A/Vietnam/3212/04) or low virulent H1N1 (A/Hong Kong/54/98) viruses in human monocyte-derived macrophages at 1-, 3-, and 6-h post infection. RESULTS: Our data reveals that two macrophage populations corresponding to M1 (classically activated) and M2 (alternatively activated) macrophage subtypes respond distinctly to H5N1 virus infection when compared to H1N1 virus or mock infection, a distinction that could not be made from previous microarray studies. When this confounding variable is considered in our statistical model, a clear set of dysregulated genes and pathways emerges specifically in H5N1 virus-infected macrophages at 6-h post infection, whilst was not found with H1N1 virus infection. Furthermore, altered expression of genes in these pathways, which have been previously implicated in viral host response, occurs specifically in the M1 subtype. We observe a significant up-regulation of genes in the RIG-I-like receptor signaling pathway. In particular, interferons, and interferon-stimulated genes are broadly affected. The negative regulators of interferon signaling, the suppressors of cytokine signaling, SOCS-1 and SOCS-3, were found to be markedly up-regulated in the initial round of H5N1 virus replication. Elevated levels of these suppressors could lead to the eventual suppression of cellular antiviral genes, contributing to pathophysiology of H5N1 virus infection. CONCLUSIONS: Our study provides important mechanistic insights into the understanding of H5N1 viral pathogenesis and the multi-faceted host immune responses. The dysregulated genes could be potential candidates as therapeutic targets for treating H5N1 disease.

Effectiveness of influenza vaccination on influenza-associated hospitalisations over time among children in Hong Kong: a test-negative case-control study.

BACKGROUND: The protection conferred by influenza vaccination is generally thought to last less than a year, necessitating annual revaccination. However, the speed with which influenza vaccine effectiveness might decline during a year is unknown, which is of particular importance for locations with year-round influenza activity. We aimed to assess how influenza vaccine effectiveness changes by time intervals between vaccination and admission to hospital, taking advantage of almost year-round circulation of influenza in Hong Kong. METHODS: In this test-negative case-control study, we analysed vaccine effectiveness in children (aged 6 months to 17 years) who were admitted to hospital in Hong Kong over 5 consecutive years (2012-17). We included those who were admitted to general wards in four public hospitals in Hong Kong with a fever (≥38°C) and any respiratory symptom, such as runny nose, cough, or sore throat. We used direct immunofluorescence assay and reverse transcription PCR to detect influenza virus infection, and recorded children's influenza immunisation history. We compared characteristics of positive cases and negative controls and examined how vaccine effectiveness changed by time between vaccination and admission to hospital with regression analyses. FINDINGS: Between Sept 1, 2012, and Aug 31, 2017, we enrolled 15 695 children hospitalised for respiratory infections, including 2500 (15·9%) who tested positive for influenza A or B and 13 195 (84·1%) who tested negative. 159 (6·4%) influenza-positive cases and 1445 (11·0%) influenza-negative cases had been vaccinated. Most vaccinations were done by December of each influenza vaccination season. Influenza-related admissions to hospital occurred year-round, with peaks in January through March in most years and a large summer peak in 2016; pooled vaccine effectiveness for children of all ages was 79% (95% CI 42-92) for September to December, 67% (57-74) for January to April, and 43% (25-57) for May to August. Vaccine effectiveness against influenza A or B was estimated as 79% (95% CI 64-88) within 0·5-2 months of vaccination, 60% (46-71) within >2-4 months, 57% (39-70) within >4-6 months, and 45% (22-61) within >6-9 months. In separate analyses by type and subtype, we estimated that vaccine effectiveness declined by 2-5 percentage points per month. INTERPRETATION: Influenza vaccine effectiveness decreased during the 9 months after vaccination in children in Hong Kong. Our findings confirm the importance of annual vaccination in children. Influenza vaccines that provide broader and longer-lasting protection are needed to provide year-round protection in regions with irregular influenza seasonality or lengthy periods of influenza activity. FUNDING: Health and Medical Research Fund, Hong Kong and the Research Grants Council, Hong Kong.