Dissertations / Theses on the topic 'Influenza, Human Influenza, Human'

Limited understanding of influenza transmission has been a frequent obstacle during the development of pandemic influenza infection prevention and mitigation strategies. The science is hotly debated, especially the relative importance of transmission via large droplets or aerosols. Clarification of the relative importance of different modes of transmission is critical for the refinement of evidence-based infection control advice and has been called for by the European Center for Disease Control (ECDC), the World Health Organization (WHO), and the US Institute of Medicine. The primary aims of this thesis were to investigate influenza transmission; i) by obtaining data concerning viral shedding and the presence of influenza virus in the near environment of infected individuals and ii) through the exploration of a human challenge model to study transmission. Two major clinical studies have been performed; • Shedding and environmental deposition of novel A (H1N1) pandemic influenza virus. The primary aims of the study were to correlate the amount of virus detected in a subject’s nose with that recovered from his/her immediate environment (on surfaces and in the air) and with symptom duration and severity. Adults and children, both in hospital and from the community, who had symptoms of influenza infection were enrolled. Information about symptoms was collected and samples were taken including nose swabs, swabs from surfaces and air samples. Forty two subjects infected with influenza A(H1N1)pdm09 were recruited and followed up. The mean duration of nasal viral shedding was 6.2 days (by PCR) and 4.6 days (by culture). Over 25% of cases remained potentially infectious for at least 5 days. Symptom scores and viral shedding were poorly correlated. From surface swabs collected in the vicinity of 40 subjects, 15 (38%) subject locations were contaminated with virus. Overall 36 of 662 (5.4%) surface swabs taken were positive for influenza, two (0.3%) yielded viable virus. Subjects yielding positive surface samples had significantly higher nasal viral loads on illness Day 3 and more prominent respiratory symptom scores. Room air was sampled in the vicinity of 12 subjects and PCR positive samples were obtained from five (42%). Particles small enough to reach the distal lung (≤4µm) were found to contain virus. • Use of a human influenza challenge model to assess person-to-person transmission: Proof-of-concept study. The primary aim of this study was to establish that an experimentally induced influenza infection is transmissible. Healthy subjects deemed sero-susceptible to influenza A/H3N2/Wisconsin/67/2005 were intranasally inoculated (Donors) and when symptoms began, further sero-susceptible subjects (Recipients) were exposed to Donors during an ‘Exposure Event’. Subjects were in close contact, e.g. playing games and eating meals together, for a total of 28 hours during a 2 day period. Samples were collected to confirm infection status. Among 24 healthy adult subjects, nine were randomised to the ‘Donor’ group and 15 to the ‘Recipient’ group. Following inoculation 5 out of 9 Donors (55%) developed illness and 7 out of 9 (78%) were proven to be infected. After exposure, 5 out of 15 Recipients developed symptoms and 3 out of 15 were proven to be infected. Three others were found to be non sero-susceptible prior to exposure. The overall attack rate in Recipients was 20% but was 25% after adjustment for pre-exposure immunity. The contact, droplet and aerosol routes of influenza transmission are all likely to have a role. This thesis shows that transmission of influenza via surfaces may be less important than current infection control policies and public guidance documents imply. Air sampling results add to the accumulating evidence that supports the potential for aerosol transmission of influenza. The human challenge model could be used to investigate routes of influenza transmission further and a study funded by the Centers for Disease Control (CDC) is planned.

Background: Early and accurate diagnosis of influenza helps start correct treatment and prevention strategies at individual level. Ongoing systematic collection, analysis and dissemination of the surveillance data from aggregated diagnostic results and other early indicators help gather the foremost disease information for all subsequent control and mitigation strategies in the community. Disease information from surveillance results then feed back to medical practitioners for improving diagnosis. By improving this loop of disease information transfer in terms of accuracy and timeliness, interventions for disease control can be applied efficiently and effectively. Methods: Several new influenza diagnosis and surveillance methods were explored and evaluated by comparing with laboratory reference test results. Logistic regression models were applied to synthesize a refined clinical guideline for human influenza infections. The performance of QuickVue rapid diagnostic test was evaluated in a community setting. Weekly positive rates from the above two diagnostic methods, together with three other different syndromic surveillance systems, including data from school absenteeism, active telephone survey and internet based survey were evaluated according to the US CDC public health surveillance systems guideline in terms of their utility, correlations and aberration detection performance. Different combinations of surveillance data streams and aberration detection algorithms were evaluated to delineate the optimal use of multi-stream influenza surveillance data. A framework of efficient surveillance data dissemination was synthesized by incorporating the merits of the online national surveillance websites and the principles of efficient data presentation and dashboard design. Results: A refined clinical diagnostic rule for influenza infection using fever, cough runny nose and clinic visit during high influenza activity months as predictors was scored the highest amount all other current clinical definitions. Time series weekly positive rate from this rule showed better correlation with reference community influenza activity than many other current clinical influenza definitions. The QuickVue rapid diagnostic test has an overall diagnostic sensitivity of 68% and specificity 96%, with an analytic sensitivity threshold of 105 to106 viral copies per ml. Weekly aggregated QuickVue and school absenteeism surveillance data was found to be highly correlated with hospital laboratory and community sentinel surveillance data, but the telephone and internet survey was only moderately correlated. Multiple univariate methods performed slightly better than multivariate methods for aberration detections in general. More sophisticated outbreak detection algorithms did not result in significant improvement of outbreak detection
published_or_final_version
Community Medicine
Doctoral
Doctor of Philosophy

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Human adaptation of avian influenza viruses pose an enormous public health challenge as the human population is predominantly naive to avian influenza antigens. As such, constant surveillance is needed to monitor the circulating avian strains. Of particular importance are strains belonging to H5N1, H7N7, H7N2 and H9N2 subtypes that continue to circulate in birds worldwide and have on occasions caused infections in humans. A key step in influenza human adaptation is the accumulation of substitutions/mutations in the viral coat glycoprotein, hemagglutinin (HA), that changes HA's binding specificity and affinity towards glycan receptors in the upper respiratory epithelia (referred to as human receptors). Unlike for the H1, H2, H3 and more recently H5 HA a correlation between the quantitative binding of HA to human receptors and respiratory droplet transmissibility has not been established for H9 and H7 subtypes. This thesis is a systematic investigation of determinants that mediate changes in HA-glycan receptor binding specificity, with focus on the molecular environments within and surrounding the glycan receptor binding site (RBS) of avian HAs, particularly the H9 and H7 subtypes. The glycan receptor binding properties of HA were studied using a combination of biochemical and molecular biology approaches including dose dependent glycan binding, human tissue staining and structural modeling. Using these complementary analyses, it is shown that molecular interactions between amino acids in and proximal to the RBS, including interactions between the RBS and the glycan receptor converge to provide high affinity binding of avian HA to human receptors. For the H9 HA [alpha]2-->6 glycan receptor-binding affinity of a mutant carrying Thr-189-->Ala amino acid change correlated with the respiratory droplet transmission in ferrets conferred by this change. Further, it was demonstrated for the first time that two specific mutations; Gln226-->Leu and Gly228-->Ser in glycan receptor-binding site of H7 HA substantially increase its binding affinity to human receptors. These approaches and findings contribute to a framework for monitoring the evolution of HA and the development of general rules that govern human adaption applicable to strains beyond ones currently under study.
by Karunya Srinivasan.
Ph.D.

The seasonal Influenza A viruses are respiratory pathogens causing epidemics annually with mild illnesses, while sporadically, novel influenza viruses emerge and trigger pandemics associated with more widespread and sometimes severe disease. The biological basis for severity of influenza disease remains unclear though it is recognized that the interplay between the influenza viruses and the host immune responses both contribute to viral pathogenesis. As macrophages are key sentinels of the innate immune response and play a crucial role in being the “first responders” as well as contributing to shaping the subsequent (pathogen‐specific) adaptive immune response, the objective of this research was to bring insights on the outcomes of the interactions of influenza viruses with the macrophages. The occurrence of Antibody‐Dependent Enhancement (ADE) of Influenza infection in macrophages was investigated. ADE occurs when non‐neutralized virus‐antibody complexes find alternative entry routes into host cells, mainly through the Fc‐receptor pathway and has been demonstrated predominantly in macrophages. Addition of human serum from some individuals to influenza A virus (either H5 pseudoparticles or pandemic (H1N1) virus) led to enhanced infection of murine macrophage‐like cells as illustrated by a two to five fold increase in detection of influenza M‐gene copies. Immunofluorescence microscopy indicated that serum‐mediated pandemic (H1N1) infection led to an increase in the number of infected cells than in controls. As the fold change in viral gene copies paralleled the fold increase of infected cells I concluded that ADE infection provide pandemic (H1N1) virus with increased opportunity to infect cells rather than simply increase the viral load per cell. In order to strengthen our results, and make them more physiologically relevant, experiments were then performed with human primary cells with clinical sera. However, ADE was not demonstrated in primary human macrophages, suggesting that ADE may be cell type or host specific. The second research question investigated was whether the different state of human primary macrophage differentiation or activation in vitro determined the susceptibility to influenza infection. Recently, work by others has shown a diverse range of macrophage phenotypes that arise by differences in macrophage differentiation and activation. In addition to the classical activation pathway (caMΦ), new mechanisms of activation, designated as alternative activation (aaMΦ), have been reported. Classically and alternatively activated macrophages display different phenotypes and properties, such as molecule expression patterns, cytokine secretion, and gene signatures. This study constitutes the first systematic comparison of Influenza A virus infection of these different subsets of human primary monocyte‐derived macrophages. When assessed for their permissiveness to different influenza A viruses, aaMΦΦshowed greater susceptibility to influenza A infection than caMΦ. This work also documents the receptor patterns and the gene expression profile of these macrophages in response to influenza virus infection in vitro. The results point to differences in susceptibility of the classically and alternatively activated human macrophages to pandemic H1N1 and other influenza A viruses and reveal intrinsic differences between these macrophage subtypes. Further investigations are needed to define the cellular and molecular determinants that define susceptibility of different macrophage subsets to influenza A infection.
published_or_final_version
Public Health
Doctoral
Doctor of Philosophy

Resumo Introdução: A Vigilância Sanitária se constitui como campo de intervenção da Saúde Pública tendo como uma de suas responsabilidades, garantir o controle sanitário de aeroportos e a proteção da saúde dos viajantes. Objetivo: Neste sentido, o presente estudo teve como objetivo conhecer, descrever e analisar a prática sanitária adotada frente à Pandemia de Influenza A (H1N1) 2009, pela Vigilância Sanitária no Terminal de Passageiros do Aeroporto de Guarulhos. Metodologia: A pesquisa qualitativa foi adotada, tendo como referencial teórico as representações sociais. Utilizou-se o referencial metodológico da hermenêutica dialética, fazendo uso de uma abordagem interpretativa reconstrutiva das falas dos entrevistados. A construção das três categorias empíricas Trabalho, Comunicação, Intersetorialidade - permitiu resgatar junto às falas dos profissionais pesquisados a prática vivenciada pela Vigilância Sanitária durante a pandemia. Resultados: Pôde-se apreender que as dificuldades evidenciadas durante a Pandemia de H1N1 estiveram relacionadas aos recursos humanos, à estrutura física e de material, ao fluxo de procedimentos e de informações. Conclusões: Os resultados evidenciaram a prática da VISA associada diretamente a sua estrutura organizacional; a uma atuação coadunada com o desenvolvimento atual do país; e uma experiência que serviu como o mais importante e único teste de enfrentamento para uma pandemia de influenza
Abstract Introduction: The Health Surveillance is a field of Public Health with the one of its responsibilities to ensure the sanitary control of airports and heath protection of travelers. Objective: In this sense, the present study aimed to understand, describe and analyze the sanitary practice adopted on the face of Influenza A (H1N1) Pandemic in 2009, by the Health Surveillance Agency in the passengers arrival gates of Guarulhos Airport. Methodology: The qualitative research was adopted in this study, using as a theoretical reference the social representations. In this document it was used the referral method of Hermeneutic Dialectic, using the interpretation of the interviews. It was built three empirics categories, which allowed retrieving in the interviews the practical experience of the employees of the Health Surveillance during the Pandemic period. Outcomes: It could be learnt that the difficulties during the A H1N1 Pandemic was related to the human resources, physical and material infrastructure and the process and information flows. Conclusion: The outcomes emphasized the way the Health Surveillance works directly linked to its organizational structure; its behavior, aligned with the current Brazil situation; and the experience which was a unique test of how they face the Influenza pandemic

Influenza A viruses cause seasonal epidemics and pandemics in the human population, resulting in significant morbidity and mortality. Influenza can be controlled by vaccination and antiviral therapy. However, antigenic drift, reducing vaccine effectiveness, and the development of antiviral resistance can result in reduced efficacy of the control measures. Drugs that target host cell processes, such as glycosylation, may be employed to complement drugs that target the virus, and iminosugar compounds which inhibit α-glucosidases have been reported to show antiviral activity against some viruses. Here, I have examined the effect of two iminosugars on human influenza A viruses. I have shown that two α-glucosidase inhibitors, N-butyl deoxynojirimycin (NB-DNJ) and N-nonyl deoxynojirimycin (NN-DNJ), show antiviral activity in cell culture against three human influenza A viruses: a recently circulating seasonal H3N2 virus strain, A/Brisbane/10/2007, an older H3N2 strain, A/Udorn/307/72, and a representative of the currently circulating pandemic H1N1 virus, A/Lviv/N6/2009. Of the two, NN-DNJ was the more potent drug. The virus target and mode of action of NN-DNJ has been examined. The effect of the drug was most marked after infection. Consistent with its action as an α-glucosidase inhibitor, NN-DNJ treatment resulted in altered glycan processing, as shown by a reduction in electrophoretic mobility of both influenza virus glycoproteins, haemagglutinin (HA) and neuraminidase (NA). NN-DNJ treatment was found to reduce cell surface expression of H3 HA. The level of sialidase activity of NA was reduced in the infected cell, however addition of exogenous sialidase to cells did not complement NN-DNJ mediated inhibition of virus replication. Using reassortant viruses, the drug susceptibility profile correlated with the HA. Reverse genetics was used to determine the effect of altering the glycosylation status of the HA; engineered viruses carrying modified sites seemed slightly more sensitive to the inhibitor than the parent virus. These results show that NN-DNJ inhibits influenza A virus replication in a strain-specific manner which is dependent on the HA.

The emergence of novel influenza A viral lineages to which there is limited or no immunity remains an on-going concern for human and animal public health. At least three of the pandemics reported in human hosts during the 20th and 21st century are believed to have emerged as a result of reassortment between human, avian and swine viruses circulating at the time. The emergence of novel lineages with pandemic potential is frequently believed to occur as a consequence of reassortment. The ability to predict when and how the next novel lineage with pandemic potential will arise remains challenging. Additionally, the dynamics of infection in systems where multiple subtypes are known to circulate remain poorly understood. Gaining insight into these biological processes is of vital importance if we are to understand how much co-infection is occurring, and hence the opportunity for reassortment. This thesis uses data analysis, novel algorithmic methods applied to genomic data and mathematical modelling to develop an understating of the dynamics of influenza A infection within non-human hosts. It seeks to analyse patterns of reported reassortment in influenza A across all hosts, geographic regions and time periods. Chapters 3 and Chapter 4 use data on reported reassortment events to identify additional, previously unreported suspected reassortant viral lineages, and develop a quantitative definition of significant reassortants. These chapters use publicly available sequence data to understand to what extent published data on reassortants reflects the possible extent of reassortment that can be detected with an iterative clustering algorithm. They develop methods that can be used to assign a rank score to suggest whether an individual isolate is a suspected reassortant. Methods presented in these chapters are applied to all publicly available influenza A sequence data from two different databases. The estimation of epidemiological parameters such as R0 and the time of virus introduction into a population (Ts) are of vital importance to infer in the event of an outbreak of a novel influenza virus n a poultry production facility. This thesis assesses the performance of different surveillance strategies to estimate R0 and Ts using simulated surveillance data from an outbreak of H7N9 in commercial poultry. Since the detection of novel H7N9, it has been isolated from numerous live bird markets. Live birds markets are known to harbour diverse subtypes of avian influenza within numerous different susceptible hosts. Co-infection with two different influenza viruses is a prerequisite for reassortment to occur therefore the final results chapter explores the conditions required to generate the multi-strain dynamics of different AI subtypes and the resulting co-infection dynamics that emerge. Taken together, through these topics addressed in this thesis we understand more broadly the possible true extent of reassortant detectable in publicly available sequence data. How surveillance data collection can be optimised to help estimate epidemiological parameters in the event of an outbreak, and the possible mechanisms that generate the multi-strain dynamics within a live bird market.

A detailed understanding of the B-cell response to influenza A haemagglutinin is key to the accurate matching of vaccines to seasonal strains, and may inform the development of broader spectrum vaccines. In this study, I develop techniques for predicting the location of the epitopes of protective antibodies by observing the physical locations of amino acid substitutions in human wild-type strains. By linking the understanding gained from this analysis with a large body of assay data, I present a model which can predict antigenic distance from HA1 amino acid sequences and which meets or exceeds the predictive power of previously developed models while retaining generality. An interesting conclusion from the epitope analysis discussed above is that antibodies to the HA head bind in two regions. The antigenic evolution of influenza H3N2 is more punctuated than its genetic evolution. I propose that the dual regions might contribute to the punctuated nature of antigenic evolution, and explore this through the use of a simple simulation. Stalk-binding antibodies to HA have attracted much interest in recent years: a number of broad-binding examples have been isolated, and the slower evolution of the stalk gives hope that these may provide broad protection against future strains. Stalk-binding neutralising antibodies to H3 are known to bind in two regions, and I use data from crystal studies to identify the constituent residues of these regions, which I term antigenic sites F and G, in a manner that is consistent with previous analyses of the constituent residues of HA1 antigenic sites A-E. I analyse the degree of conservation of residues in sites F and G, and conclude that there have been episodes of change in the H3 stalk which are consistent with antigenic evolution.

The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize
published_or_final_version
abstract
toc
Microbiology
Doctoral
Doctor of Philosophy

Understanding the epidemiology of human influenza in Viet Nam is important for developing local policies and also for understanding the dynamics of influenza in tropical and subtropical southeast Asia. I have analysed an 18 year time-series of influenza-like-illness (ILI) surveillance data, and assessed the relationship of this time-series with climate variables and with sentinel influenza virus surveillance data. I also conducted a study of influenza A/H1N1 transmission within households. ILI notifications in Viet Nam show a latitudinal gradient, with seasonality in the north but no seasonal pattern observed in low lying areas of central and southern Viet Nam. Seasonality is however observed in the elevated provinces of central Viet Nam, suggesting that the seasonal patterns are driven by climate. Principal component analysis finds that temperature and absolute humidity (AH) are positively correlated and together explain around 59% of total climatic variance, and that there is a strong latitudinal gradient in these variables. Regression tree analysis shows that provinces with strong seasonality of AH have strong ILI seasonality. Although virological surveillance data are limited, increases in ILI notifications are associated with an increase in the proportion of upper respiratory tract swabs that are influenza positive. In a prospective study of H1N1/2009 transmission in a household-based cohort, 11 of 59 household contacts were infected, giving a household secondary infection risk of 18.6% (95%CI 10.7-30.4%), but 5 (45%) did not develop symptoms. Virus genetic sequencing indicated that 10 of the 11 secondary cases (91%) were probably infected within the household rather than from the community. This research provides new insights into the seasonality and climatic determinants of ILI and influenza epidemiology in Viet Nam, and on the transmission of influenza within households. The findings are valuable for national influenza control policies and also add to the current state of knowledge of influenza epidemiology.

Objective: To investigate antiviral activity of maca to reduce viral load in kidney (MDCK) cells infected with influenza type A and B viruses (Flu-A and MFalud-inB-, Dreasrpbeyc ctiavneilny)e. Methods: Maca were extracted with methanol (1:2, v/v). The cell viability and toxicity of the eaxgtariancstts Fwluer-eA e avnaldu aFtleud- oBn v MirDusCeKs cwealsls a usssianyge dm uetshinogd aM TteTs ta sfosar yd. eAtenrtmiviinrainl ga ctthiev itiyn hoifb citoimonp oouf nthdes cytopathic effect on cell culture and multiplex RT-PCR. Results: The methanol extract of maca showed low cytotoxicity and inhibited influenza-induced cytopathic effect significantly, while viral load was reduced via inhibition of viral growth in MDCK infected cells. Maca contains potent inhibitors of Flu-A and Flu-B with a selectivity index [cytotoxic concentration 50%/IC50] of 157.4 and 110.5, respectively. Conclusions: In vitro assays demonstrated that maca has antiviral activity not only against Flu-A (like most antiviral agents) but also Flu-B viruses, providing remarkable therapeutic benefits.
Financial support of this study was provided by AECID grants (PCI: C/033641/10) and AGAUR (MAT2009-11503, MAT2012-36205, 2009SGR-1208). JDVM support was provided by 1st Concurso Incentivo a la Investigación de la Universidad Peruana de Ciencias Aplicadas, Lima, Peru.
Revisión por pares

Macrophages have well-established roles in the primary response to pathogens and hold essential functions during innate and adaptive immunity. Under activation by different growth factors and cytokines, human monocytes have been shown to differentiate and polarize into two main types of macrophage, classically-activated macrophages (caMφ) and alternatively-activated macrophages (aaMφ), displaying distinct properties and phenotypes. For instance, caMφ secrete pro-inflammatory cytokines, whereas aaM secrete anti-inflammatory cytokines. Additionally, aaMφ displays stronger phagocytic ability and are equipped with different endosomal proteases. While it has been established that monocyte-derived macrophages can be infected by Influenza A virus, most studies utilized a macrophage population obtained by differentiation in the presence of autologous plasma. My research project aimed at systematically comparing susceptibility of the infection by Influenza A virus to the recently described caMφ and aaMφ. Here I show that monocytes cultured in presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN)-γ or in presence of macrophage colony-stimulating factor (M-CSF) and interleukin (IL)-4 or IL-10 can be differentiated into distinct populations. According to immunophenotyping results, a distinct expression profile was observed for Cluster of Differentiation (CD) 36, CD86, Mannose Receptor (MR or CD206), and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209) among differentiated macrophages. Except for CD86 expression, my results were in accordance with previous reports and thus allowed me to classify all populations into caMφ (M1 macrophages), and aaMφ (M2a and M2c macrophages). I then assessed the susceptibility of the above mentioned macrophages to pandemic Influenza A/California/04/2009 H1N1 virus (CA04) infection. My results demonstrate a marked difference, caMφ showing low to moderate permissivity, whereas aaMφ – and in particular M2a macrophages – were consistently highly infected. In contrast, no difference was observed with Influenza A/WSN/1933 H1N1 virus (WSN/33) infection. Because sialic acids are regarded as the primary receptor for influenza virus, I investigated the cell surface distribution of sialic acids with α2-3 linkage (SAα-2,3) or α2-6 linkage (SAα-2,6) among the population of human macrophages. By using lectin staining with Maackia amurensis lectin (MAL) II and Sambucus nigra lectin (SNA), which bind sialic acids with α2-3 linkage (SAα-2,3) and α2-6 linkage (SAα-2,6) respectively, I found all the monocyte-derived macrophages exhibited a comparable expression of SAα-2,3 and SAα-2,6, which unlikely explain the differential susceptibility to infection by CA04. In addition to sialic acids, C-type lectins were also proposed to mediate entry of influenza viruses into macrophages. All macrophages expressed CD206 but only M2a expressed CD209. However assay aiming at interfering with CD209 binding (MAb blocking assay or EGTA treatment) did not inhibit pdmH1N1 infection. Surprisingly, infection in presence of EGTA, which is believed to reduce the functional ability of C-type lectins, exacerbated susceptibility of the macrophages. Altogether my results show that susceptibility to Influenza A virus infection of in vitro differentiated primary human macrophages is unlikely to rely on the sialic acid expression profile and is dependent on viral strain. Further studies are needed to understand what difference from caMφ and aaMφ – either phenotypic and/or biochemical – confer them distinct susceptibilities to some viral subtype/strain of Influenza A.
published_or_final_version
Pathology
Master
Master of Philosophy

Le virus de la grippe A est un virus à ARN négatif appartenant à la famille des Orthomyxoviriadea dont la réplication se produit dans le noyau des cellules infectées. L'organisation du génome est segmentée en huit segments d'ARNv de polarité négative, codant pour un minimum de 16 protéines virales différentes. Ces ARN viraux (ARNv) sont en complexe avec de nombreuses copies de nucléoprotéines et liés par leurs extrémités 5' et 3' au complexe hétérotrimérique de l'ARN-polymérase ARN-dépendante composé des sous unités PA, PB1 et PB2. Cet assemblage macromoléculaire (ARNv / polymérase / NP) nommée Ribonucléoprotéine (RNP) constitue une entité génomique indépendante. Dans le contexte de la RNP, l'ARN-polymérase assure à la fois la transcription et la réplication du génome ARNv. En assurant ces deux fonctions, l'ARN-polymérase joue un rôle majeur dans la réplication virale et constitue une cible antivirale privilégiée. Les travaux de recherche présentés dans cette thèse se concentrent sur les éléments structuraux participants à l'assemblage de l'ARN polymérase et son interaction avec les avec les ARNv. Pour atteindre ces objectifs, notre laboratoire, en collaboration avec d'autres groupes, a mis en place un système d'expression en polyprotéines permettant d'exprimer la polymérase. Plus encore, cette méthode a aussi permis de reconstituer des complexes entre l'ARN-polymérase et des partenaires cellulaires, notamment RanBP5 qui appartient à la famille des importines-β
Influenza A virus is a negative-strand RNA virus belonging to the Orthomyxoviriadea family whose replication occurs in the nucleus of infected cells. The genome organisation of influenza virus is segmented in eight vRNA segments of negative polarity coding for at least 16 different viral proteins. Each vRNA is bound to multiple copies of nucleoprotein (NP) and to the heterotrimeric RNA-dependent RNA-polymerase complex (PA, PB1 and PB2) through its 5' and 3' extremities. This macromolecular assembly (vRNA/polymerase/NP) forms the ribonucleoprotein (RNP) particle, which acts as a separate genomic entity within the virion. The RNP complex is at the core of viral replication and in the context of RNPs, the polymerase performs both transcription and replication of the vRNA genome. As such, the polymerase constitutes a major antiviral drug target. The research work presented within this thesis focuses on the underlying determinants of the RNA polymerase assembly process and its interaction with its vRNA genome. To fulfill these goals, our lab, in collaboration with other groups, has set up a novel polyprotein expression system to express the polymerase but also to reconstitute polymerase and cellular partner complexes, notably RanBP5, which belongs to the importin-β family

Complement system includes a conglomeration of a set of soluble factors and membrane anchored receptors, which has been designed to recognise and clear non self (pathogens) and altered self (apoptotic cells, necrotic cells and transformed cells). In a general theme, the recognition subcomponents bind to target, which is followed by limited proteolytic cleavage of downstream complement components. Three pathways namely classical, alternative and lectin converge on the generation of C3 convertase. The alternative pathway is activated by spontaneous cleavage of C3, generating C3a, an anaphylatoxin, and C3b, which binds to the surface of pathogens. A C3 convertase is formed, which has a half-life of about 90 seconds, is stabilised by properdin, and in an amplification loop a C5 convertase is formed leading to lytic pathway and cell lysis. This puts properdin at the heart of up regulator of alternative pathway. Properdin is structurally organised into seven thrombospondin repeats (TSR), whose functions have been delineated via deletion mutagenesis studies. In the chapter 3, we have expressed TSR4 and TSR5 in tandem in E. coli and shown that the two-module recombinant protein binds to C3b, sulfatides, and glycosaminoglycans similarly to native properdin. The recombinant module also seems to be an efficient inhibitor of properdin’s ability to stabilise C3bBb complex, thus offering a therapeutic possibility to dampen alternative pathway. Although properdin’s definite role in perpetuating the alternative pathway is well established, its structural organisation also appears to suggest its potential as an independent innate immune soluble factor that would not require engagement with complement system. In chapter 4, we report the ability of properdin to interact with mycobacterium (BCG) via TSR4+5 module, down regulate the microbial uptake by macrophages, and up regulate pro-inflammatory cytokine response via enhancing anti-mycobacterial TNF-α production. In chapter 5, we demonstrate that properdin as well as TSR4+5 interacts directly with a range of influenza A virus strains leading to inhibition of infection and dampening of pro-inflammatory response. The ability of properdin to interact with non-self is further reaffirmed by its ability to interact with nanoparticles and modulate subsequent immune cell response as presented under discussion chapter. Thus, this thesis reports a set of novel observations highlighting non-complement properties of properdin, which may be crucial in host pathogen interaction.

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

Since 2012-2013 influenza season, World Health Organization (who) recommends the formulation of tetravalent vaccines. Globally, many countries already use tetravalent vaccines in their national immunization programs, while in Latin America only a small number. Two Influenza b lineages co-circulate, their epidemiological behavior is unpredictable. On average they represent 22.6% of influenza cases and more than 50% in predominant seasons. The lack of concordance between recommended and circulating strains was 25 and 32% in the 2010-2017 and 2000-2013 seasons, respectively. There are no clinical differences between influenza A and B. It occurs more frequently from five to 19 years of age. Influenza b has a higher proportion of attributable deaths than influenza a (1.1 vs. 0.4%), or 2.65 (95% ci 1.18-5.94). A greater number of hospitalizations when the strains mismatch (46.3 vs. 28.5%; p <.0001). Different evaluations have demonstrated its cost effectiveness. The compilation of this information supports the use of quadrivalent vaccines in Latin American countries.
Revisión por pares

Orientador: Maria Rita Donalísio Cordeiro
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-26T16:23:15Z (GMT). No. of bitstreams: 1 Freitas_AndreRicardoRibasde_D.pdf: 4270845 bytes, checksum: 09078cb28a971b59a0103ccfbe6a3bee (MD5) Previous issue date: 2014
Resumo: Introdução e objetivos: As infecções respiratórias estão entre as mais importantes causas de morbimortalidade no mundo. A sua alta incidência tem relevante impacto nos óbitos, como também na sobrecarga do sistema de saúde e absenteísmo no trabalho e escola Todas as faixas etárias são acometidas, porém, as mais afetadas são as crianças e os idosos. Também são particularmente susceptíveis os imunocomprometidos e os portadores de doenças crônicas em geral. Os vírus são os agentes responsáveis pela maior parte das infecções respiratórias, os principais vírus causadores de infecções respiratórias são o influenza A e B e o Vírus Sincicial Respiratório (VSR). Estes vírus têm comportamento biológicos distintos e o conhecimento de como estes vírus afetam a saúde da população é fundamental para embasar as ações de prevenção, profilaxia e tratamento de pacientes permitindo uma alocação adequada de recursos em quantidade e tempo adequados. No Brasil, no ano 2000, para monitorar a ocorrência destes vírus foi implantada a vigilância de síndromes gripais SIVEP-GRIPE, que através de 128 unidades sentinelas distribuídas em todas as regiões do país coletam semanalmente amostras de secreção de nasofaringe por semana de pacientes com síndromes gripais. Neste trabalho estudamos o impacto do influenza na mortalidade no estado de São Paulo, nas diferentes faixas etárias no período entre 2002 e 2011, incluindo o período da pandemia de 2009. Estudamos também a sazonalidade do VSR nas 5 diferentes regiões brasileiras e o impacto deste vírus nas internações por doenças respiratórias entre menores de 5 anos. Metodologia: Para o estudo da mortalidade associada ao influenza utilizamos o método de regressão de Serfling adaptado para dados semanais extraindo da série histórica os períodos de maior circulação viral a partir dos resultados do sistema de vigilância sentinela SIVEP-GRIPE. Comparar a mortalidade associada à pandemia de influenza de 2009, às epidemias prévias anuais de influenza nas diferentes faixas etárias e com diferentes subtipos de vírus influenza circulantes no estado de São Paulo. Para o estudo da sazonalidade do VSR utilizamos análise de Wavelets, análise de Fourier, análise simplificada de estações anuais comparando os resultados nas 5 regiões administrativas do Brasil. Para identificar possíveis correlações temporais entre a circulação dos vírus respiratórios utilizamos métodos de regressão de ranque de Spearman e de regressão parcial. Resultados e conclusões: A mortalidade por pneumonia e influenza associada à pandemia de 2009 no estado de São Paulo foi ligeiramente mais alta que nos outros anos de influenza sazonal, considerando a mortalidade geral, sem distinção de faixa etária. Houve diferenças no risco de morrer entre as faixas etárias. Entre os indivíduos de 5 a 19 anos, a mortalidade associada à pandemia de 2009 foi 2,6 maior (0,6 óbitos/100.000hab) que a de anos não pandêmicos. Na faixa etária de 20 a 59 anos, a mortalidade associada à pandemia de 2009 foi 5,1 maior (2,8 óbitos/100.000hab) que nos anos não pandêmicos. As taxas de mortalidade entre menores de 5 anos foi 0,9 óbitos/100.000hab e na população de mais 60 anos foi 13,1 óbitos/100.000hab, ou seja, foram menores que nos anos não pandêmicos. O método de análise utilizado permitiu a diferenciação entre a mortalidade associada a subtipos virais (A(H3N2), B ou sazonal A(H1N1) e A(H1N1) pdm 2009). Foi possível a comparação entre a mortalidade associada à pandemia de influenza de 2009 em São Paulo, às epidemias anuais de influenza nas diferentes faixas etárias e com diferentes subtipos de vírus influenza circulando. Isto é, o impacto da circulação do vírus pandêmico influenza A(H1N1) foi maior na mortalidade em adultos e jovens, enquanto em maiores de 65 anos foi discreto. Por outro lado, o excesso de mortalidade foi expressivo em maiores de 65 anos, nos anos de circulação do influenza A(H3N2). O modelo de Serfling adaptado a dados semanais com validação por meio de dados da vigilância sentinela de síndromes gripais (SIVEP-GRIPE) mostrou-se confiável para detectar picos de maior circulação viral do Influenza e supostos reflexos na mortalidade em diferentes faixas etárias em período pandêmico, epidêmico e de circulação sazonal do vírus Influenza. Sobre o VSR foi possível identificar padrões sazonais do VSR em todas as regiões administrativas do Brasil utilizando-se dados da vigilância de síndromes gripais (SIVEP-GRIPE). Houve diferenças entre os momentos de maior circulação do vírus em algumas das cinco regiões administrativas do Brasil. Os padrões sazonais de internação por doenças sabidamente relacionadas com o VSR [Pneumonia devida a vírus respiratório sincicial, Bronquite aguda devida a vírus sincicial respiratório, Bronquiolite aguda devida a vírus sincicial respiratório, Bronquiolite (aguda, não especificada),] foram semelhantes aos encontrados pela análise da circulação do VSR por meio de dados da vigilância de síndromes gripais (SIVEP-GRIPE). Houve correlação temporal entre a circulação do VSR e as taxas de internação por doenças do aparelho respiratório em geral (Capítulo-X da CID-10) entre menores de 5 anos, nas cinco regiões administrativas do Brasil. Houve correlação temporal entre as taxas de internação entre menores de 5 anos por doenças sabidamente relacionadas com o VSR [Pneumonia devida a vírus respiratório sincicial, Bronquite aguda devida a vírus sincicial respiratório, Bronquiolite aguda devida a vírus sincicial respiratório, Bronquiolite (aguda, não especificada),] e as taxas de internação por doenças respiratórias em geral nesta faixa etária nas cinco regiões administrativas do Brasil, indicando que este é o principal vírus associado às internações de crianças até 5 anos por doenças respiratórias. De acordo com as evidências encontradas neste estudo, os esquemas de profilaxia contra o VSR hoje utilizados precisam ser revistos e particularizados para cada região do país. Entre as ações a serem revistas estão a disponibilização do palivizumabe, bem como medidas de prevenção à circulação do VSR na comunidade
Abstract: Introduction and Objectives: Respiratory infections are amongst the most important causes of morbidity and mortality worldwide. Its high incidence has significant impact on deaths, but also burdens the health system and leads to absenteeism from work and school All age groups are affected, but the most affected are children and the elderly. Are also particularly susceptible immunocompromised and patients with chronic diseases in general. Viruses are the agents responsible for most respiratory infections, the main cause of respiratory virus infections are influenza A and B and Respiratory Syncytial Virus (RSV). These viruses have distinct biological behavior and knowledge of how these viruses affect people's health is fundamental to support the prevention, prophylaxis and treatment of patients allowing an adequate allocation of resources in quantity and adequate time. In Brazil, in 2000, to monitor the occurrence of these viruses was established surveillance of influenza-like syndromes SIVEP-FLU, which through 128 sentinel units distributed in all regions of the country collect weekly samples of nasopharyngeal secretions of patients per week with influenza-like illness. In this work we study the impact of influenza on mortality in the state of São Paulo , in different age groups between 2002 and 2011 , including the period of the 2009 pandemic. We also studied the seasonality of RSV in 5 different Brazilian regions and the impact of this virus in hospitalizations for respiratory diseases among children under 5 years. Methods: To study the mortality associated with influenza used the regression method of Serfling adapted for extracting weekly data of the time series periods of increased viral movement from the results of sentinel surveillance system SIVEP - FLU . Compare the mortality associated with pandemic 2009 influenza , annual epidemics of influenza prior at different ages and with different subtypes of influenza viruses circulating in the state of São Paulo . To study the seasonality of RSV , we use wavelet analysis , Fourier analysis , simplified analysis of annual seasons comparing the results in five administrative regions of Brazil . To identify possible temporal correlations between the circulation of respiratory viruses use regression methods of Spearman rank and partial regression. Results and Conclusions: The mortality from pneumonia and influenza associated with the 2009 pandemic in the state of São Paulo was slightly higher than in the other years of seasonal influenza, considering the overall mortality, irrespective of age. There were differences in the risk of dying between age groups. Among individuals 5-19 years, the mortality rate associated with the 2009 pandemic was 2.6 higher than that of non-pandemic years. (0.6 deaths /100,000 inhabitants) In the age group 20-59 years, the rate associated with the 2009 pandemic mortality was 5.1 higher than in non-pandemic years. (2.8 deaths /100,000 inhabitants). Mortality rates among children under five years was 0.9 deaths /100,000 inhabitants and in persons over 60 years was 13.1 deaths /100,000 inhabitants, ie were lower than in non- pandemic years . The method of analysis used allowed the differentiation between mortality associated with viral subtypes (A(H3N2), A(H1N1) and B or seasonal A(H1N1) pdm 2009) . It was possible to compare the mortality associated with the 2009 influenza pandemic in Sao Paulo, annual influenza epidemics in different ages and with different subtypes of influenza viruses circulating. That is, the impact of the circulation of influenza A(H1N1) pandemic virus was higher mortality in adults and children, while in adults over 65 years was low . On the other hand, the excess mortality was significant in adults over 65 years ago, in circulating influenza A H3N2. The Serfling model adapted to weekly data validation using data from sentinel surveillance of influenza-like illness (SIVEP - GRIPE) was reliable for detecting peaks of higher viral circulation of influenza and alleged impacts on mortality in different age groups in pandemic period , epidemic and seasonal circulation of influenza viruses . About RSV was possible to identify seasonal patterns of RSV in all administrative regions of Brazil using surveillance data of influenza syndromes (SIVEP -GRIPE). There were differences between the moments of greatest circulation of the virus in some of the five administrative regions of Brazil. Seasonal patterns of hospitalization for known diseases with RSV [ Pneumonia due to respiratory syncytial virus , acute bronchitis due to respiratory syncytial virus , acute bronchiolitis due to respiratory syncytial virus bronchiolitis ( acute , unspecified ) ] were similar to those found by analysis of the movement of data through RSV surveillance of influenza-like syndromes ( SIVEP - GRIPE) . There was a temporal correlation between the circulation of RSV and the rates of hospitalization for respiratory diseases in general (Chapter X of ICD- 10) among children under 5 diseases in the five administrative regions of Brazil . There was a temporal correlation between the rates of hospitalization among children under 5 years for known diseases with RSV [ Pneumonia due to respiratory syncytial virus , acute bronchitis due to respiratory syncytial virus , acute bronchiolitis due to respiratory syncytial virus bronchiolitis ( acute , unspecified ) ] and rates of hospitalization for respiratory diseases in general in this age group in the five administrative regions of Brazil , indicating that this is the main virus associated with hospitalizations of children under 5 years due to respiratory diseases . According to the evidence found in this study , the schemes of prophylaxis against RSV used today need to be reviewed and individualized for each region of the country . Among the actions to be reviewed are the availability of palivizumab , as well as measures to prevent the circulation of RSV in the community
Doutorado
Epidemiologia
Doutor em Saude Coletiva

The influenza virus has a large impact on global health; however, it is difficult to formulate vaccines and influenza therapies that are effective against influenza. The influenza virus mutates rapidly, has the ability to emerge as novel strains with pandemic potential and can quickly become resistant to any given drug. Therefore, the generation of novel anti-influenza therapeutics that are effective against multiple strains would be highly beneficial. To date, the majority of anti-influenza research has focused on targeting specific components of the virus in order to interfere with its replication. However, it has been proposed that host proteins and signaling pathways may be essential components to viral replication and could also become novel anti-influenza drug targets. Therefore, this study utilized a large proteomic screen to identify host proteins that were up- and down-regulated in response to influenza infection. Collectively, these proteins clustered into five specific cell pathways and processes including interferon signaling, purine metabolism, cell death, ubiquitin-like signaling and mitochondrial oxidoreductases. Overall, this project identified potential novel anti-influenza targets in primary airway epithelial cells.
May 2015

Neutralising antibodies and antigen-specific B cells are important for protection against influenza A virus. However, the antigenic evolution of influenza A viruses has made a continuing challenge to the design of vaccine and the public health. The ability to generate cross-reactive response against influenza remains unclear in human. It is important to explore the antibody and B cell repertoire at single cell level. The pandemic H1N1 and seasonal influenza vaccine induced robust antibody response in adults. However, pre- or co-vaccination with the seasonal vaccine led to a significantly reduced antibody response to pandemic H1N1 virus. Whether this interference has impact on subsequent infection rates remains undetermined. There observed substantial cross-reactive antibody response upon vaccination, as measured by HI, MN and B cell ELISpot assays. The antibody recognizing conserved proteins could be the main component of cross-reactivity against influenza A strains and subtypes. A significant expansion of influenza-specific MBC was observed after infection. Crossreactive response was also noted in the MBC response. Importantly, a robust early-phase ASC response was detected in the peripheral blood upon influenza vaccination or infection. The size of ASC response significantly correlated with serum HI, MN and anti-HA IgG titre three weeks after vaccination. The sequence analysis revealed that early-phase ASC accumulated high level of somatic mutations on Ig variable region and affinity maturation, as well as anti-influenza mAb, which suggested their origin from pre-existing MBC. Eight anti-influenza mAb were made from early-phase ASC, including one high-titre virus-neutralising HA1-specific, two other HA1-specific, one cross-reactive HA2-specific, and four cross-reactive NP-specific antibodies, indicating of the broad diversity of ASC repertoire. In conclusion, this study demonstrated the properties of antibody and B cell responses to influenza A virus at serological, cellular and sequence level. The virus-neutralising and cross-reactive mAb derived from ASC could have therapeutic potential and their analysis might direct the vaccine design in the near future.

The diversity of virus-specific antibodies and of B cells among different individuals is unknown. Using single-cell cloning of antibody genes, we generated recombinant human monoclonal antibodies from influenza nucleoprotein-specific memory B cells in four adult humans with and without preceding influenza vaccination. We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes. The heavy chains (HCs) originated from 26 and the kappa light chains (LCs) from 19 different germ line genes. Matching HC and LC chains gave rise to 43 genetically distinct antibodies that bound influenza NP. The median lengths of the CDR3 of the HC, kappa and lambda LC were 14, 9 and 11 amino acids, respectively. We identified changes at 13.6% of the amino acid positions in the V gene of the antibody heavy chain, at 8.4 % in the kappa and at 10.6 % in the lambda V gene. We identified somatic insertions or deletions in 8.1% of the variable genes. We also found several small groups of clonal relatives that were highly diversified. Our findings demonstrate broadly diverse memory B cell repertoires for the influenza nucleoprotein. We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification. Thus, structurally conserved proteins can elicit broadly diverse and highly mutated B-cell responses.

Influenza A virus (IAV) is a major threat to human health with seasonal epidemics, occasional pandemics and emergence of new highly pathogenic strains from the animal reservoir. Our laboratory has shown that the human Annexin A6 (AnxA6) interacts with the IAV M2 proton channel and limits production of progeny IAV from infected cells. We have found that overexpression of AnxA6 impairs morphogenesis and release of progeny viruses. These findings are supported by another study showing that AnxA6 has a critical role in the late endosomal cholesterol balance and affects IAV replication and propagation in AnxA6-overexpressing cells. However, the molecular mechanism responsible for restriction of IAV morphogenesis by AnxA6 is still unclear. AnxA6 is a calcium-dependent phospholipid-binding protein which plays a major role in cellular events such as regulation of cholesterol homeostasis and membrane organisation or repair. AnxA6 is also implicated in the regulation of intracellular signalling pathways required for IAV infection. In this study, we used a combination of virology, cellular biology and biochemistry approaches to decipher the restriction mechanism of IAV by human AnxA6. We found that AnxA6 down-regulates M2 viral protein expression and impairs viral morphogenesis and budding. We also found that AnxA6 regulates chemokines and cytokines expression during viral infection, suggesting that AnxA6 triggers an innate immune response to IAV by modulating signalling pathways required for viral replication. Finally, we observed that IAV down-regulates AnxA6 expression at mRNA level during early stages of infection and at protein level during late infection, suggesting that IAV has developed a strategy to respond to AnxA6 restriction mechanism during viral infection. We conclude that it is essential to better understand the interaction between human AnxA6 and IAV to elucidate the potential of AnxA6 as an antiviral candidate.

This thesis describes an analysis of the B cell repertoire in humans in response to infection or vaccination with Influenza or Ebola. We isolated monoclonal antibodies (mAbs) from individuals using single B cell cloning and PCR technology, and defined their epitopes, binding characteristics and neutralisation properties. As summarised by Thomas Francis in 1960, we found that the secondary B cell response to Influenza is largely determined by the primary response to the virus that the donor was exposed to in childhood. In some individuals this can lead to focusing of the polyclonal antibody response to a single site on the influenza haemagglutinin. Monoclonal antibodies isolated from such focused responses selected escape mutations in vitro that matched actual antigenic drift observed in circulating viruses. Recapitulation of antigenic drift in vitro with human mAbs, in parallel with standard analysis with ferret anti-sera, may contribute to improved selection of vaccine strains by the WHO. A second consequence of preferential selection of B cell responses from memory cells laid down early in life, is the expansion of broadly cross-reactive clones during exposures to viruses that are only distantly related to the original stimulus. We have isolated many protective antibodies to conserved epitopes on both haemagglutinin and neuraminidase from individuals with appropriate exposure histories. One novel antibody binds to the conserved active site of neuraminidase. Antibodies of this type may have therapeutic potential to complement antibodies to the conserved stem of Haemagglutinin. In contrast to Influenza, the antibody response to the Ebola glycoprotein (GP) in vaccinated humans was essentially primary. The elicited antibodies were closer in sequence to germline than those to Influenza, and contained fewer somatic mutations. The response was more diverse, employing a wide selection of VH/L genes, and was directed at multiple epitopes in at least three distinct regions on the GP. Despite these features, half of the antibodies neutralised an Ebola surrogate virus in vitro, and many were therapeutic in a murine infection model. We developed a cocktail of antibodies to three non-overlapping sites for testing as a therapy in a stringent model of Ebola infection in the guinea pig. Taken together our results fit with a Darwinian model of selection of the fittest B cells in the germinal centre reaction, where memory cells have a selective advantage over naïve B cells.

Highly pathogenic avian influenza H5N1, which is panzootic in poultry, continues to spread and becomes endemic in Asia, Africa, and Europe. It causes human disease with high fatality (about 60%) and continues to pose a pandemic threat. The pathological lesions associated with human H5N1 disease is Acute Respiratory Distress Syndrome (ARDS). The biological basis underlying the development of ARDS in human H5N1 disease remains controversial. Clinical, animal and in vitro studies suggested that the differences between H5N1 influenza viruss and low pathogenic influenza viruses in regard to viral replication, tissue tropism and cytokine dysregulation may contribute to disease pathogenesis. We previously found delayed onset of apoptosis in influenza H5N1 virus infected human peripheral blood monocyte-derived macrophages. This may allow a longer survival time for the virus in target cells for prolonged viral replication, which may contribute to the pathogenesis of H5N1 disease. As human bronchial and alveolar epithelial cells are target cells of influenza virus, I explored if influenza H5N1 and H1N1 virus infected human respiratory epithelial cells displayed differential apoptotic response and dissected the apoptotic pathways triggered by influenza virus infection. In this study, the apoptotic response in highly pathogenic influenza H5N1 viruses, A/HK/483/97 and A/Vietnam/1203/04, their precursor avian influenza H9N2 virus, A/Quail/HK/G1/97, and seasonal H1N1 virus, A/HK/54/98 infected primary human alveolar and bronchial epithelial cells was compared by TUNEL. A delayed onset of apoptosis in influenza H5N1 viruses and avian H9N2 virus infected alveolar epithelial cells was observed; the pattern was similar in bronchial epithelial cells. Concomitantly, by Western blotting, a delay in caspase 3 activation in H5N1 virus (A/HK/483/97) infected alveolar epithelial cells compared to H1N1 virus (A/HK/54/98) infected cells was shown. Also, influenza H5N1 and H1N1 virus induced apoptosis through both intrinsic and extrinsic pathways in human alveolar epithelial cells. Chemokine IP-10 was differentially up-regulated in influenza H5N1 virus infected alveolar epithelial cells, but its relationship to the delayed onset of apoptosis requires further studies. TRAIL, an upstream signaling molecule of extrinsic apoptotic pathway, mRNA was up-regulated in influenza H5N1 infected alveolar epithelial cells but not in influenza H1N1 infected cells. Using recombinant viruses, I showed that the 627 amino acid residue on PB2 of H5N1 virus and mutation of amino acids on 253 and 591 residues on PB2 of H9N2 virus contribute to the TRAIL upregulation. Immunohistochemical staining of physiologically relevant ex vivo model of human bronchus showed that influenza H5N1 (A/Vietnam/3046/04) and H9N2 (A/Quail/HK/G1/97) virus did not infect human bronchi as well as human H1N1 (A/HK/54/98) virus. Profiling of apoptosis related genes showed that TRAIL tends to be up-regulated in H5N1 virus infected bronchi ex vivo. This study demonstrated the delayed onset of apoptosis by H5N1 virus infected respiratory epithelial cells may be a mean for influenza virus to have prolonged replication within the human respiratory tract and contribute to disease severity. The results generated provide a robust research agenda, yielding critical information that elucidate molecular mechanisms, such as TRAIL up-regulation, that may contribute to the virulence and pathogenesis in human H5N1 disease.
HKU 3 Minute Thesis Award, 2rd Runner-up (2011)
published_or_final_version
Pathology
Master
Master of Philosophy