Objectives: To identify factors that are important for parental decisions on vaccinating their children against seasonal influenza based on a modified health belief model. Study design: Cross-sectional study Subjects: A total of 325 parents who had at least one child aged between 6 months and 3 years were recruited from a women and children’s hospital in Guangzhou, China Methods: Eligible subjects were identified by doctors when parents took their children to the outpatient clinic for regular body examination. Each eligible subject was invited for a face-to-face interview based on a standardized questionnaire. Hierarchical logistic regression models were conducted to identify factors associated with parents' intention to vaccinate their children and children’s actual vaccination uptake against seasonal influenza on the basis of a modified health belief model. Results: Uptake of seasonal influenza within the preceding 12 months among the target children who aged between 6 and 36 months was 47.7%. Around 62.4% parents indicated as being “likely/very likely” to take their children for seasonal influenza vaccination in the next 12 months. The hierarchical logistic regression model showed that children’s age (OR=2.59, 95%CI: 1.43-4.68), social norm (OR=2.08, 95%CI: 1.06-4.06) and perceived control (OR=2.96, 95%CI: 1.60-5.50) were significantly and positively associated with children’s vaccination uptake within the preceding 12 months; children with a history of taking seasonal influenza vaccine (OR=2.50, 95%CI: 1.31-4.76), perceived children’s health status (OR=3.36, 95%CI: 1.68-6.74), worry/anxious about their children influenza infection (OR=2.31, 95%CI: 1.19-4.48) and perceived control (OR=3.21, 95%CI: 1.65-6.22) were positively association with parental intention to vaccinate their children in the future 12 months. However, anticipated more regret about taking children for the vaccination was associated with less likely to vaccinate children within the preceding 12 months (OR=0.21, 95%CI: 0.08-0.52). Conclusions: The modified health belief model provided a good theoretical basic for understanding factors associated with parents’ decisions on their children's vaccination against seasonal influenza. It is important to provide sufficient information related to influenza vaccination benefit and improve parents' confidence to access the seasonal influenza vaccine to promote parents' intention to vaccinate their children against seasonal influenza. Providing information cues such as advice from other parents whose children have been vaccinated to increase adherence to positive social norms would be effective to encourage seasonal influenza vaccination uptake among children. Information communication should also target on reducing anticipated regret about the negative consequence of vaccinating children. / published_or_final_version / Public Health / Master / Master of Public Health

Since there is an urgent need for development of vaccines and antiviral agents to combat influenza pandemics, this study aimed to develop influenza virus-like particles (VLPs) and aptamers targeting the virus particles as vaccine and antiviral agent candidates. Influenza VLPs containing three structural proteins of hemagglutinin (HA), neuraminidase (NA) and matrix 1 (M1) derived from influenza A/Hong Kong/01/2009 (H1N1) virus (HK/01) were constructed using a Bac-to-Bac baculovirus expression system. The expressed VLPs were purified by sucrose density gradient ultracentrifugation and characterized by Western blotting analysis and transmission electron microscopy. The immune responses and protective efficacy induced by VLPs were compared with those elicited by the clinically used Panenza vaccine in BALB/c mouse model. The results showed that two-dose vaccination with both VLP and the Panenza vaccine could confer complete protection. Single-dose vaccination with VLP could also provide 100% protection against lethal virus challenge, whereas single dose of an equal amount (based on HA content) of the Panenza vaccination just provided incomplete protection (67% survival rate) against the lethal virus challenge. Compared to the Panenza vaccination, the VLP vaccination could induce higher and broader antibody responses and higher viral specific T help (Th) cell and cytotoxic T lymphocyte (CTL) responses. Notably, a novel finding in this study is that the VLP vaccination could induce antibodies to inhibit virus release from infected MDCK cells, although the underlined mechanism needed to be further studied. These results indicated that influenza VLP might be a more effective and safe vaccine candidate which could be developed into an alternative vaccine for the control of epidemic and pandemic influenza in the future. To develop aptamers as antiviral agents against influenza, I sought to use influenza VLPs as target for ssDNA aptamer selection. After 11 rounds of selection using the systemic evolution of ligandsby exponential enrichment (SELEX),the recovered DNA molecules were PCR-amplified, gel purified and cloned into pCR-Blunt II TOPO vector for sequencing. The sequencing results showed that one aptamer Va-1 was markedly enriched, which was accounted for 59% (13/22) of the selected aptamers. Compared to the other non-enriched aptamers, the enriched aptamer Va-1 showed the highest binding affinity to the UV inactivated influenza HK/01 virus. It was also shown that the aptamer Va-1 specifically bound to the HK/01 stain while it could not bind other respiratory viruses even the PR8 strain within the H1N1 subtype. It was further demonstrated that the aptamer Va-1 could only bind to NA protein in a dose-dependent manner but not bind to HA and M1 proteins. Unfortunately, the selected aptamer did not show any antiviral effects. However, it may be potentially developed into a diagnostic and analytic agent because its binding activity was comparable with that of the commercial anti-NA antibody. In conclusion, the influenza VLPs may be a promising vaccine candidate for the control of influenza virus infection and the selected aptamer may be potentially developed into an alternative tool for influenza virus detection. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

abstract / Public Health / Doctoral / Doctor of Philosophy

Background Avian influenza A (H5N1) has been circulating around and remains to be one of the major threats to human beings since it first emerged in 1997. Besides vaccines, currently there are two major countermeasures to infection in clinical settings, which are antiviral therapies and immunomodulator treatments. Objectives To summarize evidence on the effectiveness of current treatments against H5N1 infection and to explore the potential benefits of several immunomodulatory agents. Design Systematic review of cross-sectional studies and case series. Data sources Searches of PubMed for articles using the search term “(H5N1[Title]) AND antiviral[Title/Abstract]” and also manual search on PubMed for studies that are cited in some review papers in the first automatic search. Previous search results relating to human H5N1 infection studies are also included. Study selection Included studies that were human H5N1 infection cross-sectional studies or case series in which clinical outcomes were reported, CFR and survival rate were specified or could be easily derived from original data. Results 11 articles met the selection criteria and were included in our analysis. Sample size of the included studies ranged from 8 to 308 lab confirmed cases with median age varied from 10 to 29. Leukopenia, lymphopenia, thrombocytopenia and elevated ALT and AST at admission were strongly associated with worse clinical outcomes with different significance across studies. Oseltmivir treatment was generally initiated earlier among those who survived. Survival benefit of oseltamivir was the most significant if the patient received the treatment within the first two days after symptom onset, and it is still significantly effective when treatment was given up to eight days after symptom onset according to one study we included. Corticosteroid didnot show any beneficial effect or it is associated with a higher risk of death when it is given according to the current treatment protocol and a delayed initiation time. Conclusion Oseltamivir treatment is associated with survival benefit especially when initiated within the first two days after symptom onset, while immunomodulator therapies haven’t shown such benefit so far in clinical setting but some experiments in vitro and in vivo support their use in a manner which is different from the current protocol. / published_or_final_version / Public Health / Master / Master of Public Health

Background Influenza is an infectious respiratory illness which causes not only mild illness but also severe illness and death, responsible for about 250,000 to 500,000 deaths worldwide every year. Excess mortality usually had been used to estimate the actually recorded number of deaths that exceeded the number expected on the basis of past seasonal experience so as to assess the influenza related mortality. Previous studies reporting the association with environmental factors, such as ambient temperature and influenza related mortality, varied in different cities. Therefore, the association between environmental factors and excess mortality of influenza is still controversial and inconclusive, particularly in subtropical regions. Furthermore, whether there exists heterogeneity in the influenza disease burden and effect magnitude among different cities in the same subtropic region has been seldom documented so far. Objective The primary aim of this study is to critically assess the association between environmental factors especially weather conditions such as temperature, precipitation and relative humidity and the influenza‐associated mortality via systematic review and quantative analysis. The secondary aim is to compare the effect magnitude of environmental factors on influenza associated mortality between the southern cities of Mainland China and Hong Kong. Method Because influenza disease is difficult to detect or measure, influenza associated excess mortality which includes all cause deaths associated with influenza, respiratory and cardiovascular diseases (R&C) and pneumonia and influenza (P&I) has been used to measure the influenza. Eiligible studies up to February 2013 have been searched in Pubmed, EMBASE database and reference lists of previous reviews. All observational studies including ecological studies which assessed the assoicaitons between environmental factors and influenza‐associated mortality were included. Related excess mortality which had been deal with Poisson model in Hong Kong and related excess mortality which had been deal with negative binomial model in these Mainland southern cities had been collected from the secondary data. Meteorological data in Hong Kong had been collected from the historical data in meteorological observation stations, while the meteorological data in the southern cities of China was collected from China Meteorological Data Sharing Service System. Pearson correlation and linear regression have been used to examine the association between environmental factors and influenza associated mortality. Last, a pooled analysis was conducted by including city*environmental factors (temperature) as an interaction term in the model to detect the effect magnitude in the two kinds of cities. All tests were two‐sided and p values < 0.05 were considered statistically significant. Results A total of 14 papers were included in the critic review. Negative association between environmental fctors and influenza had been reported in many studies, although inconsistent results had also been reported. The excess mortality of pneumonia and influenza disease in the five southern Mainland cities is significant higher than the the one in Hong Kong (P=0.010). No significant difference was observed in all‐cause excess mortalities and cardiovascular and respiratory disease excess mortality between the two regions (P=0.991, P=0.109). In the five Mainland southern cities, there was significant association between temperature and influenza related all‐cause excess mortality, and the significant association was also found between temperature and cardiovascular and respiratory disease excess mortality (r = ‐0.475, P=0.016 and r = ‐0.673, P=0.007, respectively). Moreover, no significant association was found between precipitation and excess mortality in Mainland. While in Hong Kong, the only two significant associations were found between environmental factors, temperature and relative humidity, and P&I excess mortality (r = ‐0.763, P=0.003 and r =‐0.804, P=0.005, respectively). In the pooled analysis, the influence of temperature on R&C and all cause excess mortality was significantly different between Hong Kong and the five southern Mainland cities. With the increasing of the temperature, the excess mortalities reduced in the two counterparts. However, tempereture had a greater effect on the excess mortality in Mainland China cities than in Hong Kong. / published_or_final_version / Public Health / Master / Master of Public Health

Influenza (IV) and pneumococcal polysaccharide vaccination (PPV) may reduce hospitalization and mortality but the effectiveness of these vaccines in older adults (≥65 years) is controversial. This thesis includes seven parts with a total of ten studies studying different aspects regarding IV and PPV in institutionalized older adults - the group with the highest infection-related morbidity and mortality. 　　 　　In Part I, we presented the controversies about effectiveness of influenza and pneumococcal vaccination in institutionalized older adults. 　　 　　In Part II, we studied a retrospective cohort of 1737 older adults showing that nursing home residence is independent risk factor of infection-related mortality and hospitalization. 　　 　　In Part III, the second and third studies were systematic reviews showing that IV and PPV could reduce pneumonia and death.. 　　 　　In Part IV, we evaluated the effectivenss of IV and PPV through prospective cohorts. The fourth study was a prospective cohort study of 1859 institutionalized older adults showing that IV significantly reduced mortality and hospitalization. The fifth study was a prospective cohort study of 532 institutionalized older adults showing that when the IV strain does not match the circulating strain, PPV provided additional protection in reducing mortality. 　　 　　In Part V, the sixth study was a randomized controlled trial of 100 institutionalized older adults showing that intradermal IV has better immunogenicity than intramuscular vaccination without compromising safety. 　　 　　In Part VI, we identified factors that may affect clinical effectiveness of IV. The seventh and eighth studies were prospective cohort studies of 711 institutionalized older adults showing that vaccine efficacy declined with increasing impaired functional status and renal function. 　　 　　In Part VII, we identified determinants of receiving IV and PPV in institutionalized older adults. The ninth study was a cross-sectional study of 155 institutionalized older adults showing that encouragement from nHCWs was a major facilitator of receiving vaccination. The tenth study was a cross-sectional study of 1300 nHCWs showing that 40.2% of nHCWs had encouraged residents to receive vaccination. 　　 　　In conclusion, ten studies from this thesis demonstrated that IV and PPV are effective in preventing hospitalization and reducing mortality in institutionalized older adults. Different strategies in improving its effectivenss and acceptance were suggested. / published_or_final_version / Medicine / Master / Doctor of Medicine

Development of effective vaccines against mutable viruses (i.e HIV-1 and influenza) remains a big challenge. Antibody-dependent cell-mediated cytotoxicity (ADCC) has been found to be a key component of immune protection against viral infections in vivo. Therefore, vaccine immunogens that elicit broadly neutralizing antibodies with high ADCC are desired for vaccine development. This study is to identify primary immunogens that can initiate somatic maturation of germline antibodies of known broadly neutralizing HIV-1 antibodies (bnAbs) for HIV vaccine development and to localize dominant ADCC epitopes on hemagglutinin (HA) of pandemic H1N1 influenza virus for development of a flu vaccine. Based on the observations that known HIV-1 bnAbs have extensive somatic mutation compared to their germline versions and that HIV-1 envelope (Env) glycoprotein lacks measurable binding to putative germline antibodies of known bnAbs, we hypothesized that non-HIV-derived (poly)peptides may serve as primary immunogens to trigger somatic maturation of germline antibodies of bnAbs, leading to elicitation of intermediate antibodies (iAbs) that can further mature to HIV-1 bnAbs upon Env vaccination or HIV-1 infection. Using b12 as a model bnAb, we identified five non-HIV-derived (poly)peptides that bound to putative b12 germline and iAbs, and immunized rabbits with the (poly)peptide priming followed by Env boosting. Rabbit immunization with (poly)peptides alone induced high titers of antibodies that were cross-reactive with gp140SF162 trimer and resurfaced Env RSC3, and the serum IgGs neutralized SF162 and JRFL. These results suggest that the (poly)peptides might structurally mimic CD4bs of Env. Priming rabbits with (poly)peptides followed by boosts with gp140SF162 and RSC3 resulted in antibodies capable of competing with b12 for binding to gp140SF162 trimer and neutralizing cross-clade isolates, while control rabbits without priming produced antibodies that were unable to compete with b12 for gp140SF162 trimer binding, and the serum IgGs neutralized only 3 clade B isolates. Our results provide proof of concept that non-HIV-derived (poly)peptides may serve as primary vaccine immunogens to initiate guided immune responses towards bnAbs. HA protein has high level of immunogenicity and considered the most important target for immune protection against influenza virus infection. Several potent HA-specific bnAbs have been reported with their conserved neutralizing epitopes revealed, but there has been no report so far about ADCC epitopes on HA. Using yeast display and flow cytometry assisted cell sorting, we mapped the epitope of convalescent plasma IgGs with different ADCC activity, we identified two dominant ADCC epitopes, designated HA-E1 [AA92-117] and HA-E2 (AA 124-159), on HA of 2009 pandemic H1N1 influenza virus. E1 and E2 overlapped with the immunodominant epitopes of HA. Depletion of purified patient plasma IgGs with yeast cells expressing E1 or E2 peptides decreased ADCC activity of the IgGs. E1 and E2 sequences are highly conserved in H1N1 strains, but less so in other subtypes of influenza A viruses. Our study may aid in designing immunogens that can elicit antibodies with high ADCC activity. Vaccine immunogens designed to include the structural determinants of potent bnAbs and ADCC epitopes may confer a comprehensive immune protection against influenza virus infection. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Interleukin-17A (IL-17A)is an important pro-inflammatory cytokine that plays a critical role in host defenses against diverse pathogens. Studies have shown that IL-17Aplays protective role against sub-lethal H1 and H3 subtypes influenza infections, but it is unclear about the role of IL-17A in the highly pathogenic H5N1 and lethal H1N1 influenza virus infection. B cell is an important effector cell type in anti-influenza immunity. Although roles of B cell in influenza infection have been extensively investigated, it is unclear whether and how IL-17AregulatesB cell response during influenza infection. I examined the role of IL-17A against influenza infection by challengingIL-17A knockout (KO) and wild-type (WT) mice with highly pathogenic H5N1 and lethal H1N1 influenza viruses. Following challenge, IL-17AKO mice exhibited significantly lower survival rate, profoundly reduced body weight, more severe tissue damage and higher viral burden in the lung tissues. These evidences suggest that IL-17Aplays a protective role in lethal influenza infection. To study whether IL-17Amodulates B cell response against influenza, I found that both B-1a and B-2 cells were detected in the lung tissue and pulmonary draining lymph node, Mediastinal lymph node (MedLN),as early as 2days post-infection. Meanwhile, B-1a cells predominantly contributed to the early virus-specific IgM in the respiratory tract. However, virus-specific IgM markedly reduced in IL-17A KO mice when compared with WT controls. Adoptive transfer of B-1a cells or B-1a cell-derived antibodies conferred protection in IL-17A KO mice. These results demonstrate that IL-17A plays a critical role in modulating early antibody production of B-1a cells against lethal influenza infection. To further elucidate how IL-17A regulates B-1a cell response, I observed that B-1a cells migrated into MedLN and lung tissues during infection and underwent plasmacytic differentiation with increased antibody production in airways. IL-17A deficiency impaired these processes of B-1a cells, while intra-nasally instillation of IL-17A restored B-1a cell response by promoting both B-1a cell migration and plasmacytic differentiation. By inducing blimp-1 expression in B-1a cells in an NF-κB dependent pathway, IL-17A directly promoted plasmacytic differentiation of B-1a cells both in vivo and in vitro. Furthermore, chromatin immuno-precipitation analysis confirmed that NF-κB directly bound to the promoter of blimp-1 gene and promoted blimp-1 expression in B-1a cells following IL-17A stimulation. To determine the functional significance of IL-17A signaling in modulating B cell response against influenza infection, I first uncovered markedly reduced B cell response, predominantly B-1a cell response in IL-17A KO mice, showing reduced local migration and impaired plasmacytic differentiation in the early stage of infection. Next, intra-nasal administration of IL-17A into IL-17A KO mice significantly restored this B-1a cell response. Moreover, I detected expression of IL-17A receptor in B-1a cells. IL-17A treatment could promote antibody production from B-1a cells by inducing blimp-1 expression in an NF-κB dependent pathway. Taken together, these findings identify a novel role of IL-17A in actingas an immune modulator of B cell response against influenza infection, which will contribute to a fuller understanding of B cell biology and anti-viral response in host defense. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Influenza A virus infection is a major cause of human morbidity and mortality. T cell immunity is believed to play critical roles for host defenses against influenza A infection. Once intracellular influenza A infection is established, viral clearance is mainly dependent on virus-specific CD8+ T cells. CD4+ T cells are important for adaptive immunity to natural influenza A infection or vaccination by providing help to B cells for antibody production and also providing help to CD8+ T cells for the generation of cytotoxicity. In addition, virusspecific CD4+ and CD8+ T cells are rich sources of effector cytokines, such as IFN-and TNF-, which can promote the function of antigen presenting cells and have direct antiviral activity. Cross-subtype reactive CD4+ and CD8+ memory T cells also affect the clearance of virus infection even in those who lack virus-specific antibodies. Therefore, the aim of our study is to assess the influenza virus-specific T cell responses and define their possible protective role in pandemic H1N1 virus and seasonal influenza infection in human. First we determined whether healthy adults have the cross-reactivity of memory CD4+ and CD8+ T cells against pandemic virus. In April of 2009, 7 pandemic H1N1 infected patients and 17 their healthy contacts who had no pandemic influenza infection were recruited in this study. By using intracellular IFN-staining and flow cytometry, we examined their pandemic H1N1 virus and seasonal influenza H1N1-specific CD4+ and CD8+ T cell responses. Healthy contacts did have measurable but low frequencies of cross-reactive influenza-specific CD4+ and CD8+ T cells, though the frequencies of these T cells specific to pandemic H1N1 virus were slightly lower than that specific to seasonal H1N1 virus. Furthermore, when compared the pandemic H1N1-specific T cell responses between healthy contacts and patients with pandemic H1N1 infection, we can found that the healthy contacts have higher pandemic H1N1 specific-T cell responses than patients, suggesting these pre-existing pandemic H1N1 specific-T cells may have protection from pandemic influenza virus infection. In addition, we conducted a prospective T cell immunity and influenza surveillance study in a cohort of more than 200 healthy volunteers before the influenza season and investigated whether the pre-existing T cell immunity is related to the protection from influenza infection in the next coming influenza season. Using intracellular IFN-staining assay, we examined their pre-existing seasonal influenza H1N1, H3N2, seasonal influenza B virus-specific CD4+ and CD8+ T cell responses. Due to the small number of cases of influenza infection in the coming influenza season, the results only showed a trend that the subjects who have higher frequency of influenza virus strain-specific T cells may have lower chance to suffer from same strain of influenza infection, which to some extent, reflect the pre-exist memory T cells have association with the protection in the coming influenza season. In conclusion, T cells play an important role in defensing against influenza infection. The higher influenza virus specific-T cells response activity in healthy adults may have a protection against influenza virus infection. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Influenza A virus is one of the most common human pathogens which caused considerable disease burdens through annual epidemics and occasional pandemics. The consequences vary from mild to severe or even fatal. What are the host and viral elements which determine the consequence of infection? In the past 15 years, several avian influenza A viruses including H5N1, H9N2, H7N7 and H7N9 subtypes were found to cross host barrier and infect humans. Question about how avian influenza A viruses gained the ability to replicate in human cells remains unanswered. Studies on host factors associated with virus replication would provide important information for understanding host restriction, virus pathogenesis and for antiviral drug development. Nuclear factor 90 (NF90) is a host protein identified in our previous study to inhibit influenza A virus replication. Antiviral activity of NF90 was also found for other viruses. However, detailed mechanisms for the antiviral function of NF90 remains largely unknown. This study is focused on NF90’s antiviral functions through exploring its relationship with PKR activation and stress granules formation using influenza A virus as a model. I characterized the interaction between NF90 and PKR, and showed the C-terminal of NF90 interacts with PKR in an RNA-binding dependent manner. Using transient and stable NF90 knockdown cells, I found that NF90 is required for PKR activation upon stimulation by dsRNA or infection with a NS1 mutated virus. PKR activation leads to the formation of stress granules and stall of protein translation. I found that NF90 is a core component of stress granules, which may underlie the mechanism for the antiviral activity of NF90. However, NF90 may also complete with PKR for RNA binding and regulate PKR activation. To further delineate the interaction between NF90 and PKR by using influenza A virus, my study constructed a panel of NS1 mutant viruses which were attenuated in antagonizing specific host antiviral pathways. I characterized the NS1 123-127 mutant virus which is unable to inhibit PKR phosphorylation but retained other functions unaffected. It was demonstrated that NF90 mediates PKR-dependent antiviral pathway since NS1 123-127 mutant virus replicated to a comparable level as wild type virus in the NF90 knockdown but not scramble knockdown 293T cells or in the interferon deficient Vero cells. This study for the first time found NF90 serves as a regulator of PKR antiviral pathway. To understand the mechanism for NF90 inhibition of influenza A virus replication, I found that NP, but not the other polymerase subunits, of influenza A virus was targeted to the stress granules. Since NF90 interacts with NP, it is reasonable to postulate that NF90 mediates the localization of NP, and possibly viral mRNA, to the stress granules in order to inhibit influenza A virus replication through regulation of proteins synthesis. In summary, my study provided comprehensive evidence to support a novel NF90-PKR antiviral pathway and suggests that NF90 may play critical roles to balance PKR phosphorylation in response to virus infection in cells. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy