The influenza A virus is responsible for 250,000 to 500,000 deaths every year worldwide and millions more could die in the event of a serious pandemic. Vaccines against influenza have existed for long, but until today they have been limited by extensive production times and reduced cross-protection between different strains of the virus. This leads to a recurrent need to update the vaccine composition every year, which is both costly and inadequate to fight pandemics. An innovative approach that could improve the vaccine efficacy has been recently developed based on the selection of conserved influenza epitopes with potential to induce broader immune responses. The 23-amino acid extracellular domain of the M2 protein (M2e) is highly conserved among different influenza A strains and thus it seems like an ideal candidate for a universal influenza vaccine. However, due to its small size, it is a poor immunogen when used on its own. The aim of this project was to produce M2e-presenting virus-like particles (VLPs) in Nicotiana benthamiana plants via Agrobacterium-mediated transient expression. Plants are increasingly being examined as alternative recombinant protein expression systems due to their safety, scalability and rapid production times. Moreover, numerous studies suggest the use of recombinant virus-like particles (VLPs) to increase the immunogenicity of antigens. Therefore, to obtain VLPs presenting the M2e epitope, I genetically engineered several different M2e-HA fusion proteins by replacing the hemagglutinin (HA) globular head and main epitope with five tandem repeats of M2e epitope sequences (5xM2e) from human, swine, and avian origin influenza A viruses. To increase the chances of obtaining VLPs, M2e-HA fusions either contained the HA stalk domain (5xM2e-HAstalk) or simply the transmembrane region (5xM2e-HAtrans). Furthermore, the tetramerizing leucine zipper derived from the General Control Protein (GCN4) was also included in some of the constructs to promote particle formation. In total, six different M2e-HA fusions were created: 5xM2e-GCN4-HAstalk, 5xM2e-GCN4-HAtrans, 5xM2e-HAstalk, 5xM2e-HAtrans, 1xM2e-HAstalk and 1xM2e-HAtrans. The expression of these proteins was optimized in plants by testing different conditions and using three different expression vectors. Overall, I was able to show expression after only 3 days post-infiltration for most of the M2e-HA v fusion proteins utilizing the pEAQ-HT and pRIC 3.0 expression vectors whereas expression levels with pTRAc were low or non-detectable. Once the expression of the M2e-HA fusions was optimized, the two proteins with the highest potential to form VLPs were selected for further characterization (5xM2e-HAstalk and 5xM2eHAtrans). Using transmission electron microscopy to analyse purified proteins, both 5xM2eHAstalk and 5xM2e-HAtrans were shown to assemble into VLPs resembling the shape and size of native HA VLPs. These VLPs could also be observed in the apoplastic fractions of infiltrated leaves. However, due to the low number of particles observed, the successful incorporation of the M2e peptide on the surface of the particles was inconclusive, as shown by M2e-specific immuno-gold labelling experiments. Furthermore, contrarily to previous studies, co-expression of the M2e-HA fusions with the M1 protein resulted in a decrease in recombinant protein accumulation and VLP formation in our plant system. A possible inhibition mechanism by the M1 protein is discussed. In summary, this research provides preliminary data to produce universal influenza vaccines in plants. I report here for the first time that M2e fused to either the stalk or transmembrane domain of the HA protein, can self-assemble into VLPs without any other proteins, in N. benthamiana plants. Future work on the immunogenicity of the VLPs produced in this study is required to confirm their potential as a universal influenza vaccine that can be rapidly produced.

Influenza A virus (IAV) is a significant problem worldwide, and respiratory disease is further complicated by secondary bacterial infection. The emergence of highly pathogenic strains of IAV in conjunction with the increase of antibiotic-resistant bacteria threatens human health. A large-animal model effective for study of polymicrobial infection comparable to humans must therefore be developed. IAV has been studied extensively in small animals, including mice, rats and ferrets. However, these species frequently require IAV adaptation, reducing the capacity of these models to adequately represent human infection. Furthermore, species commonly used lack likeness to humans in both the presentation of symptoms and in lethality of infection. However, pigs are naturally susceptible to unadapted IAV and are considered to be the 'mixing vessel' for the recent pandemic IAV virus. Pigs are also susceptible to infection with Staphylococcus aureus, the most commonly isolated bacteria from IAV-infected human adults. Therefore, the use of pigs in the study of polymicrobial respiratory infections would be ideal for characterizing a host immune response comparable to humans, as well as for the development of diagnostics and therapeutics. Using this novel model, we determined that pigs are susceptible to Staphylococcus aureus, swine IAV, and polymicrobial infection. Furthermore, we showed that IAV infection predisposes pigs to Staphylococcus aureus pneumonia, and this susceptibility is dependent on day post-IAV infection. / Master of Science

Resuspended floor dust constitutes up to sixty percent of the total particulate matter in indoor air. This fraction may also include virus-laden particles that settle on the floor after being emitted by an infected individual. This research focuses on predicting the concentration of influenza A viruses in resuspended dust, generated by people walking in a room, at various heights above the floor. Using a sonic anemometer, we measured the velocity field from floor to ceiling at 10-cm intervals to estimate the magnitude of turbulence generated by walking. The resulting eddy diffusion coefficients varied between 0.06 m2 s-1 and 0.20 m2 s-1 and were maximal at ~0.75-1 m above the floor, approximately the height of the swinging hand. We used these coefficients in an atmospheric transport model to predict virus concentrations as a function of the carrier particle size and height in the room. Results indicate that the concentration of resuspended viruses at 1 m above the floor is about seven times the concentration at 2 m. Thus, shorter people may be exposed to higher concentrations of pathogens in resuspended dust indoors. This study illuminates the possibility that particle resuspension could be a mode of disease transmission. It also emphasizes the importance of considering resuspension of particulate matter when designing ventilation systems and flooring in hospitals and residences. / Master of Science

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

The interferon (IFN) system forms an essential part of the innate immune response, up-regulating hundreds of IFN-stimulated genes (ISGs) in response to viral infection. A key protein in this response is the human myxovirus resistance protein MxA, an IFN-induced GTPase with broad-spectrum antiviral activity, capable of inhibiting many RNA and DNA viruses. One of the most studied antiviral effects of MxA is the inhibition of influenza A virus replication, yet the molecular mechanism of antiviral activity is still unknown. Influenza A viruses are inhibited by MxA at two distinct stages of viral replication; during viral entry and following primary transcription of viral mRNAs. The antiviral effects of MxA during viral entry are highly dependent on IFN, however activity exerted after primary transcription can occur in the absence of IFN. This study provides evidence that MxA exerts its antiviral activity at these two stages of viral replication through distinct mechanisms, and outlines a potential model of MxA antiviral activity following primary transcription. A potential third antiviral mechanism of MxA is proposed based on the findings that MxA is able to regulate cellular lipid metabolism, thereby potentially affecting virion composition. Mutational analysis of MxA highlights the significance of GTPase activity to the antiviral effects of MxA, while also demonstrating that natural single nucleotide polymorphisms in MxA have the potential to severely impair or prevent antiviral activity. Finally, this thesis shows for the first time that MxA exhibits antiviral activity against influenza B viruses. Overall this thesis provides new information illustrating how MxA provides potent antiviral activity against influenza viruses. Such information is vitally important as understanding the molecular basis of how proteins such as MxA function against many human pathogens is fundamentally important in our efforts to create better long-term treatment options for all viral diseases.

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