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.

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.

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.

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.

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.

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.

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.

Generation and characterization of influenza A viruses with altered polymerase fidelity.

Genetic diversity of influenza A viruses (IAV) acquired through the error-prone RNA-dependent RNA polymerase (RdRP) or through genetic reassortment enables perpetuation of IAV in humans through epidemics or pandemics. Here, to assess the biological significance of genetic diversity acquired through RdRP, we characterize an IAV fidelity variant derived from passaging a seasonal H3N2 virus in the presence of ribavirin, a purine analogue that increases guanosine-to-adenosine mutations. We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses. The H5N1 PB1-V43I-recombinant virus replicates to comparable titres as the wild-type virus in vitro or in the mouse lungs. However, a decrease in viral population diversity at day 3 post inoculation is associated with a tenfold reduced lethality and neurotropism in mice. Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis.

Conservation of T cell epitopes between seasonal influenza viruses and the novel influenza A H7N9 virus.

A novel avian influenza A (H7N9) virus recently emerged in the Yangtze River delta and caused diseases, often severe, in over 130 people. This H7N9 virus appeared to infect humans with greater ease than previous avian influenza virus subtypes such as H5N1 and H9N2. While there are other potential explanations for this large number of human infections with an avian influenza virus, we investigated whether a lack of conserved T-cell epitopes between endemic H1N1 and H3N2 influenza viruses and the novel H7N9 virus contributes to this observation. Here we demonstrate that a number of T cell epitopes are conserved between endemic H1N1 and H3N2 viruses and H7N9 virus. Most of these conserved epitopes are from viral internal proteins. The extent of conservation between endemic human seasonal influenza and avian influenza H7N9 was comparable to that with the highly pathogenic avian influenza H5N1. Thus, the ease of inter-species transmission of H7N9 viruses (compared with avian H5N1 viruses) cannot be attributed to the lack of conservation of such T cell epitopes. On the contrary, our findings predict significant T-cell based cross-reactions in the human population to the novel H7N9 virus. Our findings also have implications for H7N9 virus vaccine design.

Aerosol transmission is an important mode of influenza A virus spread.

Influenza A viruses are believed to spread between humans through contact, large respiratory droplets and small particle droplet nuclei (aerosols), but the relative importance of each of these modes of transmission is unclear. Volunteer studies suggest that infections via aerosol transmission may have a higher risk of febrile illness. Here we apply a mathematical model to data from randomized controlled trials of hand hygiene and surgical face masks in Hong Kong and Bangkok households. In these particular environments, inferences on the relative importance of modes of transmission are facilitated by information on the timing of secondary infections and apparent differences in clinical presentation of secondary infections resulting from aerosol transmission. We find that aerosol transmission accounts for approximately half of all transmission events. This implies that measures to reduce transmission by contact or large droplets may not be sufficient to control influenza A virus transmission in households.

Experimental challenge of chicken vaccinated with commercially available H5 vaccines reveals loss of protection to some highly pathogenic avian influenza H5N1 strains circulating in Hong Kong/China.

Highly pathogenic avian influenza (HPAI) H5N1 virus continues to circulate in poultry in Asia and Africa posing a threat to both public and animal health. Vaccination, used as an adjunct to improved bio-security and stamping-out policies, contributed to protecting poultry in Hong Kong from HPAI H5N1 infection in 2004-2008 although the virus was repeatedly detected in dead wild birds. The detection of clade 2.3.4 H5N1 viruses in poultry markets and a farm in Hong Kong in 2008 raised the question whether this virus has changed to evade protection from the H5 vaccines in use. We tested the efficacy of three commercial vaccines (Nobilis, Poulvac and Harbin Re-5 vaccine) in specific pathogen free white leghorn chickens against a challenge with A/chicken/Hong Kong/8825-2/2008 (clade 2.3.4) isolated from vaccinated poultry in Hong Kong and A/chicken/Hong Kong/782/2009 (clade 2.3.2). Harbin Re5 vaccine provided the best, albeit not complete protection against challenge with the clade 2.3.4 virus. All three vaccines provided good protection from death and significantly reduced virus shedding following challenge with the clade 2.3.2 virus. Only Harbin Re-5 was able to completely protect chickens from virus shedding as well as mortality. Sera from vaccinated chickens had lower geometric hemagglutination inhibition titers against A/chicken/Hong Kong/8825-2/08, as compared to two other clade 2.3.4 and one clade 0 virus. Alignment of amino-acid sequences of the haemagglutinin of A/chicken/Hong Kong/8825-2/08 and the other H5 viruses revealed several mutations in positions including 69, 71, 83, 95, 133,140, 162, 183, 189, 194 and 270 (H5 numbering) which may correlate with loss of vaccine protection. Our results indicated that the tested HPAI H5N1 (2.3.4) virus has undergone antigenic changes that allow it to evade immunity from poultry vaccines. This highlights the need for continued surveillance and monitoring of vaccine induced immunity, with experimental vaccine challenge studies being done where indicated.

Use of ex vivo and in vitro cultures of the human respiratory tract to study the tropism and host responses of highly pathogenic avian influenza A (H5N1) and other influenza viruses.

The tropism of influenza viruses for the human respiratory tract is a key determinant of host-range, and consequently, of pathogenesis and transmission. Insights can be obtained from clinical and autopsy studies of human disease and relevant animal models. Ex vivo cultures of the human respiratory tract and in vitro cultures of primary human cells can provide complementary information provided they are physiologically comparable in relevant characteristics to human tissues in vivo, e.g. virus receptor distribution, state of differentiation. We review different experimental models for their physiological relevance and summarize available data using these cultures in relation to highly pathogenic avian influenza H5N1, in comparison where relevant, with other influenza viruses. Transformed continuous cell-lines often differ in important ways to the corresponding tissues in vivo. The state of differentiation of primary human cells (respiratory epithelium, macrophages) can markedly affect virus tropism and host responses. Ex vivo cultures of human respiratory tissues provide a close resemblance to tissues in vivo and may be used to risk assess animal viruses for pandemic threat. Physiological factors (age, inflammation) can markedly affect virus receptor expression and virus tropism. Taken together with data from clinical studies on infected humans and relevant animal models, data from ex vivo and in vitro cultures of human tissues and cells can provide insights into virus transmission and pathogenesis and may provide understanding that leads to novel therapeutic interventions.

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.

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.