Severe influenza virus infection, including human infection with highly pathogenic H5N1 viruses is characterised by massive pulmonary inflammation, immunopathology and excessive cytokine production, a process in which macrophages may play a vital role. The aim of this project was to investigate the hypothesis that inhibition of inflammatory responses from infected macrophages, using either alternatively activated bone marrow derived macrophages (BMDMf), or IFNg receptor deficient (IFNgR-/-) mice may ameliorate the devastating immunopathology and inflammation routinely observed in highly pathogenic influenza virus infections. Infection of alternatively activated BMDMf resulted in enhanced positivity for viral proteins, compared with classically activated, inflammatory BMDMf. However, neither subset propagated the infection indicating that while infection is abortive in both classical and alternatively activated BMDMf, the latter may prove more efficient at removing infectious virus from the site of infection due to enhanced infectivity. However, influenza virus was capable of driving expression of proinflammatory mediators such as iNOS and TNFa from classical and alternatively activated BMDMf even in the absence of IFNg signalling. IFNgR-/- BMDMf demonstrated a reduced inflammatory response to infection compared to Sv129 counterparts, suggesting a potentially impaired inflammatory response in vivo. This was investigated by infection of IFNgR-/- mice, which resulted in ameliorated disease, lower viral titres and mild immunopathology, demonstrating that inhibition of IFNg signalling limits the severity of disease. Additionally, mRNA expression for key inflammatory mediators was reduced, demonstrating that inhibition of the overwhelming inflammatory response to influenza virus infection is beneficial to the host, resulting in protection from immunopathology and improved prognosis, without impairing viral clearance.

Influenza A virus (family Orthomyxoviridae) is one of the most important human pathogens, causing annual epidemics with significant worldwide mortality, and sporadic but potentially devastating pandemics. The influenza A viral genome encodes 14 proteins and consists of 8 segments of negative-stranded RNA. During infection, the virus exploits the host cell signaling machinery to ensure efficient replication. The PI3K/Akt and Ras/ERK are two of the signaling cascades that are induced for virus survival. Influenza A virus replicates in the nucleus, hence the newly synthesized RNPs must be exported from the nucleus and exported to the cell membrane. Although the detailed mechanism of vRNP nuclear export is not yet fully elucidated, several studies on this process have begun to emerge. Influenza A virus nucleoprotein nuclear export is CRM1-dependent. Ran-binding protein 3 (RanBP3) is a Ran-interacting protein that is best known for its role as a cofactor of CRM1-mediated cargo nuclear export. In this study, we investigated the role of RanBP3 during the influenza A virus life cycle. We found that RanBP3 was phosphorylated at Ser58 in early and late phases of infection. Knockdown of RanBP3 expression led to a vRNP nuclear retention, suggesting that RanBP3 is involved in vRNP nuclear export. Moreover, we demonstrated that RanBP3’s function during vRNP nuclear export is regulated by phosphorylation at Ser58, and the RanBP3 phosphorylation is modulated by both PI3K/Akt and Ras/ERK/RSK pathways in the late phase of viral infection. In conclusion, this study has shown that RanBP3 is a host factor that has a vital role during the late stage of influenza A virus replication, specifically as a co-factor in CRM1-mediated nuclear export. Identifying this host factor will contribute to the understanding of the mechanism of vRNP transport.

The 1918 “Spanish Flu” pandemic was cause by an Influenza A virus that infected 500 million people and nearly 50 million people died. Influenza viruses utilize a RNA-Dependant RNA Polymerase (RdRp) complex, composed of the PA, PB1 and PB2 proteins along with the viral nucleoprotein (NP) to mediate viral transcription and replication. The 1918 PB1 gene has been linked to increased virulence in mice and ferrets. We have investigated the role of PB1 in the transcription and replication of the 1918 virus and its relation to pathogenicity by comparing its RdRp to the RdRp of a low virulence conventional H1N1 human virus isolate, A/Canada/RV733/2007 (RV733). Contrary to previous studies, our dual-luciferase reporter assay revealed the 1918 RdRp had lower transcriptional activity than the RV733 RdRp in vitro. The 1918 NP seems to be the key determinant for the difference in transcriptional activity of the 1918 and RV733 RdRp complexes. The 1918 PB1 in the RV733 RdRp maintained high reporter expression while the RV733 PB1 in the 1918 RdRp abolished reporter expression. 1918/RV733 chimeric PB1 proteins were also generated and evaluated with the reporter assay. Recombinant RV733/1918 viruses were generated by reverse genetics and we determined that PB1 is a key determinant of the high growth phenotype of the 1918 virus, but only a minor contributor to pathogenicity. A novel role for the 1918 NP in the high growth phenotype and pathogenicity of the 1918 virus is also described.

Avian influenza H5N1 causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. NP is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymophcyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physiochemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.

The antigenic variability of the Influenza virus hinders our ability to develop new therapeutic and vaccine strategies which provide a broad protection against all influenza strains. It has been previously suggested that a means to approach this challenge is to identify conserved sequences within viral proteins and use these for future therapeutic targets. Although such conserved sequences are plentiful amongst the internal viral proteins, their lack of exposure to the host immune system makes mounting an immune response against these regions difficult. Alternatively, the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) have been shown to provide host protection against a limited number of influenza strains when used as vaccine targets; however conserved regions within these proteins which are also antibody accessible are extremely rare. My Ph.D. thesis project is focused on investigating the functional role of a conserved region within the NA protein and to further determine the protection afforded by a monoclonal antibody to this region. In a comprehensive bioinformatics analysis, the only universally conserved sequence amongst all influenza A and B viral NA has been previously identified as being located between amino acids (a.a.) 222-230 (dubbed the HCA-2 region). However, the potential role of this region remains largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics and growth kinetics, I have found that substitutions in this sequence significantly affect viral replication by impairing the catalytic activity, substrate-binding and thermostability of NA. These findings prompted me to further investigate if antibody to this region may provide protection against influenza infection. Indeed, universal monoclonal antibody (HCA-2 MAb) against this peptide provided broad inhibition against all nine subtypes of NA in vitro and heterosubtypic protection in mice challenged with lethal doses of mouse-adapted viruses. I further demonstrated that residues within this peptide that are exposed on the surface of NA and located in close proximity to the active site, I222 and E227, are indispensable for antibody-mediated inhibition. These data are the first to demonstrate a monoclonal antibody against the NA protein which provides heterosubtypic protection. Since I observed that the HCA-2 antibody provided a broad inhibition against all nine subtypes of influenza A NA, I decided to investigate whether this inhibitory effect could be extended against Influenza B. Here, I have further reported that HCA-2 MAb provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. I also demonstrate that the growth and NA enzymatic activity of two drug resistant influenza B strains are also inhibited by the HCA-2 antibody. The findings of my Ph.D. thesis project have thus demonstrated that the HCA-2 region is paramount to optimal viral function. Additionally, my data show that antibodies generated against this region provide heterosubtypic protection both in vitro and in vivo and against drug resistant strains. These results indicate that this universally conserved epitope should be further explored as a potential target for future antiviral intervention and vaccine-induced immune responses.

A fetus’ in utero environment has a profound effect on the individual’s development in postnatal life. Research has suggested that intrauterine growth restricted children have a less robust response to vaccination. Studies have confirmed similar results in animal models; however, the effect of low birth weight on clinical disease expression is unclear. This research aims to determine if pigs from low birth weight litters have increased severity of disease after experimental infection with influenza A virus (IAV) when compared to their counterparts from high birth weight litters, thus clarifying the effect of litter birth weight on disease expression. Pilot trials were conducted to determine the appropriate dose of virus to use and the optimal days post inoculation for necropsy to use for the main trial. The results indicated that the main trial should use an inoculation dose of 1 x 107 plaque forming units of IAV and the time of necropsy should be 48 hours post inoculation. In the main trial, male piglets (n=68) from parity one or two sows were identified at farrowing as coming from high or low birth weight litters. At four weeks of age, intratracheal IAV inoculation was performed (day 0) and pigs were euthanized at 48 hours post inoculation. Clinical signs were assessed prior to euthanasia. After euthanasia macroscopic and microscopic lesion severity were assessed, along with immunohistochemical staining intensity of IAV in lung tissue. SearchLight Chemiluminescent Array Technology was used to measure the concentration of the inflammatory cytokines interleukin 1 beta, interleukin 6, and interleukin 8 in bronchoalveolar lavage fluid. Interferon alpha was measured using fluorescent microsphere immunoassay. Fifty Percent Tissue Culture Infective Dose was used to measure influenza viral titers in lung tissue. The study found no differences in clinical scores or cytokine concentration between pigs from high and low birth weight litters. Gross, histopathological and immunohistochemical scores were significantly higher in piglets from high birth weight litters and viral titers trended higher in these piglets. These findings indicate that pathologic disease scores in piglets experimentally inoculated with IAV are more severe in piglets from high birth weight litters.

Human cytotoxic T lymphocytes (CTL) recognize antigen in the form of short peptide fragments presented by Human Leukocyte Antigen (HLA) class 1 molecules. The HLA B27-restricted CTL response to Influenza A virus is directed against a single nine amino acid epitope within the viral nucleoprotein NP383-391. Influenza A-specific CTL lines and clones, generated from the peripheral blood of three unrelated individuals, exhibited remarkable fine specificity for NP383-391, failing to recognize synthetic variants containing single amino acid substitutions at positions 1, 4, 7 or 8. The sequences of rearranged T cell receptor (TCR) alpha and beta chain genes showed remarkable conservation of variable and joining gene segment usage, and also of non germline-encoded B chain residues proposed to interact with peptide, even between clones from unrelated individuals. Correlation of fine specificity for peptide with TCR alpha chain usage suggested a possible orientation for the TCR/HLA/peptide ternary complex in this immune response. The finding of restricted TCR usage in the HLA B27-restricted CTL response to influenza A virus has implications for the study and potential therapy of HLA B27 associated autoimmune disease. The TCR heterodimer from one clone was expressed in a rat basophil cell line and shown to confer specificity for HLA B27 + NP383-391. Influenza A-specific CTL were also shown to recognize bacterial superantigens in an MHC class II dependent but unrestricted manner. A rapid screening assay for candidate superantigens was developed and used to identify a novel superantigen produced by Clostridium perfringens. This superantigen was found to have a unique specificity for human TCRβ chains.

Influenza is the infectious agent of the seasonal flu. Flu symptoms from influenza infection are similar to the symptoms caused by bacterial upper respiratory tract infections. This similarity causes the inappropriate diagnosis and prescription of antibiotics, leading to drug resistant bacterial strains. Moreover, the limitations of the current viral detection methods prevent the clinical diagnosis of influenza. The objective of this project is to design a rapid and sensitive influenza diagnostic method based on the highly sensitive Nanostructured microelectrode biosensing assay. The diagnostic method was designed by selecting probe sequences, controlling the quality of the probes and the sensing chips, and optimizing the deposition conditions. This diagnostic method was shown to be capable of differentiating influenza sequences from non-complementary sequences, detecting influenza sequences in the form of ~1000-nucleotide RNA molecules, sensing the target influenza RNA within a complex mixture of cell lysates, and achieving a clinically relevant detection limit.

Influenza is the infectious agent of the seasonal flu. Flu symptoms from influenza infection are similar to the symptoms caused by bacterial upper respiratory tract infections. This similarity causes the inappropriate diagnosis and prescription of antibiotics, leading to drug resistant bacterial strains. Moreover, the limitations of the current viral detection methods prevent the clinical diagnosis of influenza. The objective of this project is to design a rapid and sensitive influenza diagnostic method based on the highly sensitive Nanostructured microelectrode biosensing assay. The diagnostic method was designed by selecting probe sequences, controlling the quality of the probes and the sensing chips, and optimizing the deposition conditions. This diagnostic method was shown to be capable of differentiating influenza sequences from non-complementary sequences, detecting influenza sequences in the form of ~1000-nucleotide RNA molecules, sensing the target influenza RNA within a complex mixture of cell lysates, and achieving a clinically relevant detection limit.

Vaccination is one of the major strategies available for combating viral infections in humans. However, currently available vaccines are not without pitfalls; they are laborious to produce, could potentially be unsafe, and in the case of the highly variable influenza virus need to be reformulated each season. The use of synthetic peptides thus represents an exciting alternative to traditional vaccines. However, these synthetic peptides are not highly immunogenic without the use of potent adjuvants. The lack of immunogenicity might be addressed by conjugation between T or B cell epitopes with universal or immunodominant T-helper epitopes. The construction of branched peptides, lipidated peptides, or designs combining both of these elements might also enhance the immunogenicity, as they might target Toll-like receptors and/or mimic the 3-dimensional structure of epitopes within the native protein. In this study, a recognized T-B peptide based on the hemagglutinin protein of the A/Puerto Rico/8/34 influenza virus was chosen as a backbone and modified to evaluate if the construction of branched peptides, lipidation, the addition of cysteine residues, or mutations could indeed alter reactivity. Screening the different designs with various antibody binding and cellular assays revealed that combining a branched design with the addition of lipid moieties leads to a greatly enhanced activity as compared to other similar T-B diepitope constructs.