Microbiology & Immunology - Theses
Permanent URI for this collection
Search Results
1 - 6 of 6
-
ItemThe epidemiology of influenza in Northern Australia( 2021)The epidemiology of influenza in Northern Australia may be unique due to the tropical climate, large Indigenous population and wide dispersal of the population in the region. To mitigate the impact of the next influenza pandemic and seasonal influenza epidemics, it is important that the epidemiology of influenza in this area be better understood. Furthermore, estimates of the effectiveness of the influenza vaccine against hospitalisation in Northern Australia and comparisons of vaccine effectiveness in Indigenous and non-Indigenous Australians are lacking. This is despite the Australian Federal Government currently covering the cost of influenza vaccination for all Indigenous Australians aged over 6 months. Chapter 2 of this thesis consists of epidemiological analyses of notified influenza cases in the Northern Territory. Rates of influenza were higher for Indigenous Australians in all age groups through 2007-2016 with the disparity being largest for those in the 55-64 age bracket (rate ratio: 5.56; 95% CI: 4.71, 6.57). Bimodal peaks in influenza activity were seen in the Top End region of the Northern Territory in 3 out of the 10 years studied. Chapter 3 details the first ever use of phylogenetic methods to describe influenza activity in the Northern Territory. Influenza strains in the Northern Territory were shown to undergo regular extinction and are related to strains present in many diverse global regions. A mismatch was seen in the influenza vaccine strain and a circulating influenza B strain during an outbreak in late 2013-early 2014. Chapter 4 consisted of a case-control study employing a test-negative design to examine the effectiveness of the influenza vaccine against hospitalisation in the Northern Territory between 2009-2014 using 1075 cases and 3461 controls. Odds ratios for vaccination in each year were obtained from logistic regression models and meta-analysed using a random-effects model. Overall vaccine effectiveness was estimated at 32% (95% CI: -1%, 54%). Vaccine effectiveness was estimated at 40% (95% CI: -10%, 68%) for non-Indigenous Australians and 23% for Indigenous Australians (95% CI: -16%, 48%). Differences in vaccine effectiveness between Indigenous and non-Indigenous Australians were not statistically significant (p>0.15), but available sample size limited ability to detect a difference. This thesis highlights the burden of influenza upon the Indigenous population of the Northern Territory, the challenges that semi-annual influenza epidemics present in this region, ongoing cycles of importation and extinction of influenza viruses occurring in the Northern Territory and provides evidence that current vaccines have limited effectiveness against hospitalisation in this area. This thesis provides a framework for examining the impact of many infectious diseases in Northern Australia.
-
ItemInvestigating the ability of RIG-I agonists to provide protection in mouse and ferret models of respiratory virus infection( 2021)Respiratory infections caused by influenza A virus (IAV) or respiratory syncytial virus (RSV) lead to substantial morbidity and mortality. Treatment options are limited and there is urgent need for the development of efficient therapeutic and prophylactic treatments. Pattern recognition receptors (PRRs) such as the cytoplasmic helicase retinoic-acid-inducible gene I (RIG-I) are part of the innate immune system. RIG-I can be activated by recognition of viral nucleic acids, leading to downstream activation of interferon-stimulated genes (ISGs) and restriction of viral replication. We have used synthetic RNA oligonucleotides to stimulate RIG-I to inhibit replication of respiratory viruses using in vitro and in vivo models of infection. Our in vitro approaches used airway cell lines from humans, mice and ferrets and investigated the effects of RIG-I agonist pre-treatment on subsequent infection with either IAV or RSV. Prophylactic RIG-I agonist treatment induced multiple ISGs and inhibited infection and growth of respiratory viruses in cell lines from each of the different species. In vivo, we utilised mouse and ferret models to study the antiviral potential of RIG-I agonists against IAV and RSV. In mice, we compared animals which do or do not express a functional Mx1 protein and found that a single prophylactic treatment with RIG-I agonist via the intravenous route resulted in ISG induction in the lungs and this correlated with reduced IAV replication. Of interest, these effects were particularly potent and long-lasting in mice expressing a functional Mx1 confirming an important role of Mx1 for RIG-I agonist-induced protection against IAV. In a mouse model of RSV, we found that a single prophylactic treatment with RIG-I agonist resulted in reduced replication of virus in the lung, as observed using bioluminescence imaging of luciferase-labelled RSV as well as plaque assay for infectious virus. Thus, our studies in mouse models indicate that a single pre-treatment with RIG-I agonists resulted in potent inhibition of two very different respiratory viruses. In ferrets, after establishing assays to monitor ISG induction in the blood and in airway tissues, we confirmed that a single intravenous injection of RIG-I agonist induced ISG induction in both peripheral blood mononuclear cells (PBMCs) and the lungs. Moreover, a single treatment prior to infection also resulted in reduced replication of both IAV and RSV in ferret lungs, although this treatment had only negligible effects on virus replication in the nasal tissues. A single treatment to animals with an established IAV infection also resulted in reduced virus titres in the lungs, suggesting its potential as a therapeutic antiviral agent. Myxoma (Mx) proteins are ISGs with potent antiviral effects against IAV. While human and mouse Mx proteins have been studied in detail, ferret Mx proteins have not been characterised. Therefore, we generated different experimental approaches to assess the induction of three endogenous ferret Mx (two splice variants of Mx1 as well as Mx2) in a ferret cell line, as well as in vitro overexpression systems to assess the cellular localisation and antiviral functions of each ferret Mx. Our findings indicate that each ferret Mx localises to the cytoplasm and that particular proteins exhibit antiviral functions against IAV, but not RSV. However, further studies are required to clearly define the antiviral activity of ferret Mx, since our preliminary results indicate that ferret Mx proteins display different antiviral activity following overexpression in human or in ferret cells. Together, studies described in this thesis demonstrate the potential of RIG-I agonists as antiviral treatments against diverse respiratory viruses both in vitro and in vivo and represent an important step towards the development of novel antiviral treatments in humans.
-
ItemNanoparticle interactions with the immune system( 2021)Vaccination has been an incredibly successful public health intervention, saving the lives of 2-3 million people each year. Despite this success, we still lack effective vaccines for many infectious diseases including HIV, tuberculosis and malaria. Nanoparticles (ordered structures within the range of 10-1000nm) have great potential to supplement traditional vaccines based upon pathogen subunits, or killed or attenuated microorganisms, as demonstrated by the successful licensure of virus-like particle vaccines for human papillomavirus and liposomal mRNA vaccines for SARS-CoV2. However, the immunological mechanisms that explain the potent immunity of nanoparticle vaccines and the factors dictating their interaction with the immune system are poorly defined. This thesis studies how nanoparticle characteristics affect their interaction with the immune system with a view to improving vaccine strategies. First, the contribution of the protein corona on the association of engineered nanoparticles with primary human blood cells was assessed. The association of high protein binding (high-fouling) mesoporous silica (MS) particles and low-fouling zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) particles with human white blood cells was assessed by flow cytometry in the presence or absence of plasma proteins. The effect of precoating nanoparticles with serum albumin, IgG and complement protein C1q was also assessed. The differential association of low and high-fouling nanoparticles was found to be largely a consequence of the de novo formed, not pre-adsorbed, biomolecular corona. Specifically, an enrichment of complement proteins within the corona resulted in an increased association with B cells. Second, the immune mechanisms that give rise to the improved immunogenicity of a prototypic nanoparticle vaccine were investigated. Humoral immune responses to a self-assembling protein nanoparticle vaccine for influenza (HA-ferritin) were contrasted to a subunit influenza vaccine (soluble HA) in mouse and non-human primate models. Antibody titres and protective efficacy of the vaccines were compared followed by a detailed study of lymph node germinal centre B cell and T follicular helper cell responses. Vaccination of C57BL/6 mice with HA-ferritin nanoparticles elicited higher serum IgG titres and greater protection against experimental influenza challenge compared with soluble HA vaccination. Within the antigen-draining lymph nodes, germinal centre reactions were expanded and persistent following HA-ferritin vaccination. This augmented humoral immunity was not driven by ferritin-specific T follicular helper cells but rather driven by expanded antigen colocalization with follicular dendritic cells. However, this immune enhancement did not translate from mice to pigtail macaques where antibody titres and lymph node immunity following HA-ferritin nanoparticle vaccination were comparable to soluble HA protein vaccination. And thirdly, we explored innate immune activation by HA-ferritin and soluble HA in mice. This was achieved through in vitro assessment of antigen glycosylation and complement activation and in vivo through serum IgM titres and cell trafficking to the lymph nodes following vaccination. HA-ferritin vaccination of mice was found to elicit an early enhancement of antigen-specific serum IgM however in vitro complement activation was not detected. Trafficking of immune cells to the lymph nodes was found to be influenced by antigen glycan composition in conjunction with purification methods. The findings of this thesis suggest that nanoparticle interaction with the immune system is driven by the complex interplay of nanoparticle physiochemical properties, antigen glycosylation, corona formation and pattern-recognition receptors of innate immune cells. Further improvements in understanding the relationship between these features and how they may differ between animal species will speed the rational design of next-generation nanoparticle vaccines against diverse pathogens.
-
ItemGeneration of protective immunity against severe influenza virus infection in Indigenous Australians( 2020)Morbidity and mortality rates from seasonal and pandemic influenza virus infections occur disproportionately in high-risk groups, including Indigenous people globally. Although adaptive immunity is essential for combating viral infections, it is largely understudied in Indigenous populations, including Aboriginal Australians. To rationally design a protective vaccine against both seasonal and pandemic influenza viruses for both Indigenous and non-Indigenous people, in-depth knowledge about B cell and T cell responses is needed. Therefore, the aim of this PhD thesis was to identify and characterise CD8+ T cell responses restricted by HLA alleles dominant in Indigenous Australians as well as understand CD4+ T cell, B cell and humoral responses to the inactivated influenza vaccine in Indigenous and non-Indigenous Australians. The data generated in this PhD thesis provide key insights into a rational design of a cross-protective influenza vaccine. The HLA-A*24:02 allele was associated with a higher mortality during the 2009 influenza pandemic and is highly expressed in Indigenous populations globally. In Chapter 3, we identified novel influenza A and B virus (IAV and IBV, respectively) CD8+ T cell targets presented by HLA-A*24:02 and characterised their immunogenicity in HLA-A*24:02 transgenic mice, and HLA-A*24:02+ Indigenous and non-Indigenous individuals. We discovered the first IBV-derived CD8+ T cell epitopes presented by HLA-A*24:02. A total of 6 novel immunogenic IBV epitopes were identified, with one epitope, A24/PB2550-558 being cross-reactive between IBV and the IAV variant, making it especially important for a cross-protective universal vaccine. We defined epitope-specific CD8+ T cells ex vivo in the blood of healthy Indigenous, non-Indigenous and acutely-infected donors. These newly identified epitope-specific CD8+ T cells were readily detected during acute infection, mainly of an effector memory phenotype and expressed activation markers such as PD-1, CD38, HLA-DR and CD71, thus highlighting their involvement during influenza virus infection. Sub-cutaneous vaccination of transgenic mice with three immunogenic IBV peptides resulted in reduced viral titres, reduced pro-inflammatory cytokines MIP-1a, MIP-1b and RANTES in the lung as well as reduced weight loss in comparison to unvaccinated mice. These data highlight the importance of identifying novel CD8+ T cell epitopes for their potential use as a universal influenza vaccine in the context of a highly prevalent HLA allele in Indigenous people globally. Indigenous Australians express a unique HLA class I profile which includes high frequencies of HLA-A*24:02, A*11:01, A*34:01, B*13:01 and B*15:21. Except for HLA-A*24:02 and HLA-A*11:01, no influenza virus-derived epitopes have been identified for the Indigenous-associated HLAs. In Chapter 4, we generated single HLA-expressing cell lines to use as antigen-presenting cells for identifying and characterising CD8+ T cell responses towards the Indigenous-associated HLAs. We optimised an influenza virus infection assay, instead of using peptide pool cultures, to stimulate and expand rare influenza-specific CD8+ T cells, and thus allow sufficient numbers for the sequential dissection of single epitope candidates. As such, we identified the first IAV and IBV epitopes presented by HLA-B*13:01. While responses to IAV were dominated by the highly variable B13/NP404-412 epitope, IBV-responses were directed towards a variety of epitopes, including the dominant and highly conserved B13/HA371-379 and B13/HA427-435 peptides. We sequenced IAV- and IBV-specific CD8+ T cell receptors and identified unique TCR signatures between the different epitopes. While B13/NP404-412-specific CD8+ T cells showed a limited bias for the expression of TRBV19 with a public TCR, B13/HA371-379-specific CD8+ T cells almost uniquely expressed TRAV3 paired with a variety of different TCRb-chains. The developed tools proved to be an efficient method to identify and characterise IAV and IBV epitopes presented by Indigenous-associated HLAs to further develop a universal influenza vaccine that can protect Indigenous Australians. Current influenza inactivated vaccine strategies delivered intramuscularly induce mainly B cell and antibody responses. Responses to the influenza vaccine are studied extensively but not fully understood in high-risk populations. Despite the strong recommendations for annual influenza vaccination, Indigenous Australians are completely underrepresented in studies that analyse vaccine responses. In Chapter 5, we defined cellular and humoral responses to the inactivated influenza vaccine in Indigenous Australians vaccinated between 2016 and 2018, and compared these responses to non-Indigenous Australians. We identified robust antibody responses to the vaccine that were comparable to non-Indigenous donors and were cross-reactive with viruses circulating more than 10 years ago. Antibody responses on day 23+ post vaccination correlated with acute activation of circulating T follicular helper cells type 1 (cTFH1) in line with previous studies. System serology of selected Indigenous and non-Indigenous donors revealed significant reduction of the contribution of IgG3 to influenza-specific antibodies in Indigenous Australians, which correlated with a higher frequency of the G3m21 allotype. The generated data are important to support vaccine recommendations for Indigenous Australians, but also highlight the need to improve influenza vaccinations by harnessing the protective capacity of CD8+ T cells in future vaccine designs. Overall, this PhD thesis provides highly important knowledge of T and B cell immunity to IAV and IBV in Indigenous Australians. The findings of this PhD thesis provide key insights into the development of a universal influenza vaccine that also protects Indigenous Australians, one of the high-risk groups of developing severe influenza disease.
-
ItemDynamics and control of T follicular helper cell-dependent and -independent responses to influenza virus infection and immunization( 2020)Seasonal influenza viruses circulate globally and cause recurrent disease in humans. Worldwide, annual epidemics are estimated to cause 1 billion infections, with 3 to 5 million cases of severe illness and 290,000 to 650,000 deaths. Influenza viruses undergo rapid antigenic evolution allowing mutant viruses to escape from host immune responses acquired to parental virus strains. Current seasonal influenza vaccines are effective when vaccine strains are matched with circulating strains. However, there is little to no cross-protection against antigenic variants, emerging pandemic or zoonotic outbreak strains. There is therefore tremendous interest in the development of novel universal vaccines which induce potent, broad and durable antibody responses against most or all influenza viruses. T follicular helper cells are crucial for the generation of high affinity antibodies and the maintenance of B cell memory. But relatively little is known about Tfh in important animal models of influenza. Insights gained from the study of Tfh cell responses will facilitate the design of next generation vaccines against influenza. In this thesis, we first developed an activation-induced marker assay for the identification of antigen specific Tfh cells in mice after influenza virus infection and hemagglutinin protein immunization. We showed that the AIM assay was robust and sensitive for the detection of murine Ag specific Tfh cells by quantifying the upregulation of surface CD154 or CD25 OX40 following either HA peptide pool or whole HA protein stimulation for 18 hours. This murine AIM assay makes it feasible to delineate Ag specific Tfh cells in mice without the need for transgenic mice or MHC II tetramers restricted to specific epitopes. Importantly, Ag specific Tfh cells can be sorted for TCR sequencing or adoptive transfer since AIM assay is a live cell assay. Ferrets are a well established animal model for influenza research and are widely used to investigate the pathogenesis and transmission of influenza viruses and preclinically evaluate the efficacy of influenza vaccines. However, little is known about ferret Tfh cells due to the lack of ferret reactive immunological reagents. To enable the study of ferret Tfh cells, we screened commercial markers of Tfh cells, antiBCL6, CXCR5 and PD1 antibodies, and found two anti-BCL6 antibodies had cross reactivity with lymph node cells from influenza infected ferrets. We also developed two murine monoclonal antibodies against ferret CXCR5 and PD1 using a single B cell PCR based method. We were able to clearly identify Tfh cells in LNs from influenza infected ferrets using these antibodies. The development of ferret Tfh marker antibodies and the identification of ferret Tfh cells will facilitate the assessment of vaccine induced Tfh responses in the ferret model and the design of novel vaccines against influenza infection. HA stem is an attractive target for the development of universal influenza vaccines due to its relatively conserved feature. However, HA stem is poorly immunogenic when administered alone in a soluble form. Immunogen multimerization can enhance the immunogenicity of poor immunogens even in the absence of the help of T cells, which serves as an alternative pathway to improve the immunogenicity of stem without the dependence on Tfh responses. We showed that chemically coupling a peptide derived from the head domain of PR8 HA, P35, with the weakly immunogenic HA stem protein caused aggregation of the HA stem which significantly enhanced stem specific B cell responses independent of Tfh cell help in mice. P35 conjugation represents a new pathway to boost stem specific antibody responses without introducing exotic carrier proteins which will elicit anti carrier responses. Collectively, we investigated Tfh responses to influenza virus infection and immunization in mice and ferrets and explored the effects of immunogen multimerization on humoral immunity in the context limiting Tfh responses to HA stem. An increased understanding of Tfh dependent and independent mechanisms to enhance humoral immune responses will assist developing novel vaccines to prevent the infection of influenza and other viruses.
-
ItemAdvancing the ferret as an immunological model to study B-cell responses( 2020)Introduction Influenza is a clinically significant disease, causing 24000-62000 deaths alone in the United States during the 2019-2020 season. While annual vaccines are available, variable efficacies have been reported and annual updates are required due to antigenic drift. Ferrets are a useful model for studying human respiratory viruses and have been widely used to evaluate vaccines and transmission of influenza. Sera from ferrets infected with different influenza strains are used in HI assays as part of strain determination of seasonal influenza vaccines. A key limitation of the ferret model is the paucity of immunological reagents to characterise immune responses and a lack of knowledge regarding the ferret immune system. This PhD thesis aims to advance the ferret as an immunological model to study human respiratory viruses by developing methods and reagents which will enable in-depth interrogation of ferret B-cell responses. Methods While a draft copy of the ferret genome is available, immunoglobulin sequence information is not well-annotated. Hence, we first annotated the ferret genome with immunoglobulin variable, diversity, joining and constant chain genes by inferring homology using human and canine orthologs (Chapter 3). Novel PCR primers targeting 5’- leader, 3’- joining and 3’- constant chain immunoglobulin genes were derived, enabling the recovery of functional, paired heavy and light chain transcript sequences from single sorted ferret B-cells. Ferret immunoglobulin constant sequences were validated by RNA-seq, which enabled the development of ferret IgG expression plasmids. Using this technique, HA-specific B-cell responses were characterised for the first time in ferrets at the transcript level (Chapter 4). Candidate ferret mAbs were derived from the recovered sequences, expressed and screened for HA binding specificity and in-vitro influenza virus neutralisation activity. We noted poor recovery of ferret HA specific mAbs and subsequently sought to improve flow cytometric panels available for ferrets. We established a methodology using previously developed murine single-cell BCR sequencing methods to recover murine anti-ferret mAbs (Chapter 5). First, coding sequences of ferret B and NK-cell reagents were identified on the ferret genome and validated by sequence and structural comparisons with other mammalian homologs. C57BL/6 mice were subsequently immunised with these antigens and candidate mAbs were recovered for examination by ELISA and flow cytometry. Results Ferret variable, diversity, joining and constant chain coding genes were identified on the draft copy of the ferret genome and show good sequence similarity to human and canine variants. Our novel ferret immunoglobulin specific PCR primers enabled the recovery and characterisation of germline ferret immunoglobulin genes from single sorted ferret B-cells. RNA-seq validation of ferret immunoglobulin constant chain genes subsequently enabled the construction of ferret IgG/IgL expression plasmids. This facilitated the expression of chimeric human-ferret CR9114 IgG antibody retaining HA binding specificity. Subsequently, using previously developed trimeric HA probes, clonally expanded sequences were recovered from single sorted HA-specific B-cells derived from infected ferrets. Screening of candidate ferret monoclonal antibodies enabled the identification of two novel antibodies, belonging to the same clonal family showing HA binding specificities. Further examination by HAI and microneutralization assays revealed the ability of the mAbs to neutralise influenza virus in vitro. Viral escape mapping revealed binding epitope to previously reported Sa site of the HA head domain, showing proof of concept for mapping HA epitopes using these recombinant ferret mAbs. We next attempted to improve flow cytometric panels for ferrets which will enhance recovery of ferret immunoglobulin transcripts. As there are currently no mAbs targeting B and NK-cell markers in ferrets, we identified key markers for murine mAb development including CD19, IgD, CD138, NKp46 and LAMP-1. We identified candidate anti-ferret CD19 and IgD mAbs which bound to cognate recombinant antigens by ELISA, validating this method for generating anti-ferret mAbs to improve panels for flow cytometry and confocal microscopy. As the mAbs in this thesis lacked the capacity to resolve ferret cell populations by flow cytometry, we identified and discussed key steps in the process which will inform future use of this approach to develop anti-ferret mAb reagents. Conclusion The body of work presented in this thesis forms the proof of concept of studying antigen-specific B-cell responses at the mAb level in ferrets. Future improvements in tools developed in this thesis and future development of reagents will enable detailed interrogation of the ferret immune system.