Microbiology & Immunology - Theses
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Dynamics and control of T follicular helper cell-dependent and -independent responses to influenza virus infection and immunization
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.
The biofilm formation and iron acquisition systems of Klebsiella pneumoniae
Klebsiella pneumoniae is an opportunistic bacterial pathogen and a common cause of healthcare-associated infections. Due to the emergence of antimicrobial-resistant and hypervirulent strains, K. pneumoniae is recognised as a major public health threat. The ability of some K. pneumoniae strains to form biofilms increases this concern, particularly in healthcare settings where bacteria can colonise surfaces of indwelling devices. Biofilms can mediate more effective host colonisation and bacterial cells within biofilms are often more resistant to antimicrobial treatments. Given the importance of biofilms in enhancing virulence and complicating treatment regimens, greater understanding of biofilm formation among Klebsiella isolates is required. This dissertation revealed that the K. pneumoniae clinical isolates AJ094, AJ097 and AJ218 exhibited different biofilm formation capabilities, and their biofilms had variable responses to DNase and proteinase treatment, suggesting qualitative differences. The early-stage biofilms formed by AJ094 and AJ218 could be destabilised by DNase, suggesting that the presence of extracellular DNA in the biofilm matrix was important for biofilm development. Dispersal of AJ218 biofilms by proteinase treatment indicated the importance of protein components such as fimbriae in maintaining the biofilm. Proteomic analysis revealed that cells were more metabolically active in the planktonic compared to the biofilm state, and differential expression of certain proteins suggested physiological variation between planktonic and biofilm cells. Proteomic experiments also showed that type 3 fimbrial proteins were expressed at higher levels in the biofilm state, particularly in the AJ218 strain, which is known to form biofilms via type 3 fimbriae. Some proteins expressed in the biofilm state were involved in metal ion uptake and AJ094, AJ097 and AJ218 were all shown to require iron supplementation for optimal growth and biofilm formation in minimal media. Mutation of an enterobactin-mediated iron acquisition gene in AJ094 significantly reduced biofilms under iron limiting conditions compared to iron-replete conditions. On the other hand, the siderophore yersiniabactin was shown to be less important than enterobactin for supporting in vitro biofilm formation and growth. Highly invasive Klebsiella strains often carry genes for yersiniabactin synthesis and, compared to enterobactin, this siderophore is less well-understood. A transposon mutagenesis approach was employed in this study to identify novel regulators and efflux systems for yersiniabactin. The study was successful in identifying known yersiniabactin-related factors, including genes located outside of the yersiniabactin operon that were directly or indirectly related to yersiniabactin metabolism and transport. During construction of the transposon mutant library, a spontaneous 70-kb chromosomal deletion occurred in the parent strain. The deletion mutant exhibited reduced siderophore activity when associated with a transposon insertion within a hypothetical gene (orf684). However, when the 70-kb region was present, inactivation of the orf684 gene no longer caused reduced siderophore activity. This study has improved our understanding of biofilm composition and the requirement of iron and siderophores in the formation of K. pneumoniae biofilms. Further research into fundamental characteristics of iron, siderophores and matrix components in regard to bacterial growth and biofilm formation may lead to the development of novel drugs or preventive strategies to reduce the burden of bacterial infection.
Characterisation of Neutralising and Functional Antibody Responses to Different HIV-1 Env Vaccines in Bovines
Two main challenges have impeded the development of an effective HIV-1 envelope (Env) vaccine, with antibodies eliciting neutralisation of virions as well as Fc-effector functions, such as antibody-dependent cytotoxicity (ADCC), phagocytosis (ADP) or complement deposition (ADCD). On one hand, designing the right Env vaccine to elicit humoral or cellular protection has been challenging and, to date, SOSIP-Env trimers which are covalently constrained in the closed, pre-fusion conformation are the best vaccine candidate over uncleaved (Unc), open-structured trimers. On the other hand, eliciting heterologous neutralising antibodies in several animal models (including humans) has been difficult. Cows nevertheless produce unique antibodies with long CDRH3 regions, capable of accessing neutralising epitopes beneath the glycan shield, inaccessible for other animals. We tested how differences in clade and/or structure of HIV-1 Env vaccines affect the neutralising activity and Fc-effector functions of antibodies elicited, using recombinant trimers of clades A (KNH1,BG505), B (AD8, PSC89) and C (MW), which exposed either an open structure (Unc gp140) or a closed structure (SOSIP gp140). KNH1/BG505 SOSIP gp140 vaccine elicited the best neutralising IgGs against heterologous tier-2 pseudoviruses with high potency and breadth. While AD8 Unc gp140 also induced neutralisation, it was against only tier-1 pseudoviruses. Nevertheless, it was the only vaccine able to elicit IgGs that engaged CD32 (FcgRIIa), induced phagocytosis and complement-activation. The different antibody profile observed with both vaccines was explained by the Env immunogen structure, as KNH1/BG505 SOSIP gp140 induced mostly IgGs targeting the V1/V2 loop, whereas AD8 Unc gp140 induced antibodies targeting CD4-binding site and CD4-induced epitopes. In addition, analysis of IgG repertoires from animals of KNH1/BG505 SOSIP 100 and AD8 Unc 500 groups showed that KNH1/BG505 SOSIP gp140 induced higher rates of somatic hypermutation in germline genes compared to AD8 Unc gp140, with each animal presenting a unique antibody profile, and with germline antibodies already presenting high affinity towards HIV-1 Env trimers, as high levels of affinity maturation were not required to obtain antibodies with high neutralising activity. Overall, the results in this work show that open structured trimers elicit antibodies which highly activate antibody-effector functions, while SOSIP trimers focus antibody responses to concealed neutralising epitopes. The high neutralising responses observed in bovines against HIV-1 Env are due to antibodies which do not need high levels of somatic hypermutations and, in particular for KNH1/BG505 SOSIP, this antigen induced high levels of affinity maturation, probably favouring the improvement of both binding and neutralisation. Our study suggests that an effective vaccine regimen may include both uncleaved gp140 and SOSIP gp140, in order to target epitopes required for antibody-dependent effector functions as well as neutralisation, or a new trimeric structure with flexibility in the gp120-gp41 interface, exposing both epitopes involved in Fc-effector functions as well as neutralising ones.
Inducing immunity to liver stage malaria through endogenous tissue resident memory cells
Tissue resident memory CD8 T (TRM) cells provide effective tissue surveillance and can respond rapidly to infection due to their strategic location. Within the liver, TRM cells can induce effective protection against liver-stage Plasmodium infection. Recently, members from our group identified a highly immunogenic peptide (named Pb 1) within the putative 60S ribosomal protein L6 of P. berghei ANKA. Experiments conducted and presented in this thesis aimed to assess the suitability of Pb 1 for the induction of endogenous liver TRM cells that confer sterilizing protection in B6 mice. To this end, a series of different immunisation strategies targeting the Pb 1 epitope were implemented and specific CD8 T cell responses were assessed. Results revealed that the number of naive specific CD8 T cell precursors for the Pb 1 epitope was very large. Substantial expansion and formation of specific liver TRM cells was achieved by two different immunisation strategies: i) Single injection with Clec9A mAb plus adjuvant and ii) Prime and trap, both targeting the Pb 1 epitope. While mice vaccinated with Clec9A mAb developed partial protection, almost all mice vaccinated with prime-and-trap targeting Pb 1 were sterilely protected against liver stage challenge. Inflammation favours the formation TRM cells and adjuvants can affect their numbers. Accordingly, a second focus of this thesis sought to investigate how to enhance liver TRM cell formation by using TLR and RIG I like receptors agonists as adjuvants. For this, eight different agonists were assessed for the generation of liver TRM cells induced by Clec9A targeted immunisation with the Pb 1 epitope. Data from this screen showed that CpG based adjuvants were most effective at inducing the formation of TRM cells in the livers of vaccinated mice and that the transfection reagent DOTAP enhanced this effect. Based on this understanding, we then investigated the potential of CpG and its encapsulation in DOTAP to improve TRM cell generation by other vaccination strategies. Surprisingly, these studies revealed that CpG based adjuvants did not improve liver TRM cell generation by vaccination with radiation attenuated sporozoites. The basis for this outcome is discussed. Altogether, these findings provide insights into elements that favour the generation of protective liver TRM cells; information that can be used for the design of TRM cell based subunit vaccines against Plasmodium infection.
Legionella pneumophila: from amoeba to macrophage metabolism
Legionella pneumophila is an aquatic bacterium that has emerged as an accidental human pathogen. Within the aquatic environment, L. pneumophila has evolved virulence factors to survive predation by environmental amoebae. These virulence factors are hypothesised to allow the adaptation of the bacteria to replicate in human alveolar macrophages. During infection, L. pneumophila forms a replicative vacuole termed the Legionella-containing vacuole (LCV) that sustains the bacterial intracellular replication. Establishment of the LCV requires the Dot/Icm type IV secretion system (T4SS), that secretes over 330 bacterial proteins termed effectors into the infected host cell in order to manipulate host processes and facilitate bacterial replication. Despite their central role in LCV biogenesis, to date most effector proteins remain uncharacterised. Therefore, to aid in the characterisation of Dot/Icm effector proteins, in this study, we generated large genomic region mutants. To date, we have created nine genomic deletion mutants (A-I), as well as two multiple-region deletion mutants (FGHI and DFGHI) in L. pneumophila, resulting in the deletion of 68 effector genes and 138 non-effector genes. These mutants were then used to identify the genomic regions important for bacterial replication in vitro and in vivo. Despite the loss of up to 42 effector-encoding genes, all mutants can replicate efficiently in THP1 macrophages. Meanwhile, in the protozoan host, at least six mutants showed a severe replication defect. Interestingly, in the mouse model, four mutants displayed an unexpected increase in bacterial burden, while one mutant showed a reduction in bacterial replication. Surprisingly, two of the mutants showing an increase in bacterial load in the mouse model were unable to replicate in Acanthamoeba castellanii. Together, these highlight the difference in requirements to survive in different hosts. This also suggests that the large effector repertoire of the Dot/Icm T4SS effectors likely evolved to enable an intracellular lifestyle in a diverse range of hosts. Finally, the mutants were also used to identify a Dot/Icm effector protein that degraded host GAPDH mRNA. RNA sequencing of infected cells revealed that L. pneumophila downregulated multiple host glycolytic mRNAs which depended on a particular Dot/Icm effector. Taken together, this project has used mutants carrying large genomic deletions to identify genetic regions important for bacterial replication, as well as those manipulating host immune defence.
Functional and cellular heterogeneity of the myeloid cell system
Cells of the myeloid lineage form the innate part of the immune system and are characterized by a high level of functional plasticity, which is required to address the diverse set of functions of these mononuclear cells. Monocytes, Macrophages and dendritic cells (DC) are collectively categorized as the mononuclear phagocyte system (MPS), to highlight their functional equipment that specializes them to the phagocytosis of pathogens as a starting point to elicit an immune response. Besides this role, cells of the MPS are also involved in a wide variety of homeostatic functions including early development and regulation of physiological processes. However, the multitude of mechanisms required to acquire this functional plasticity remains poorly understood. The work that has been performed in the scope of this dissertation aimed to advance current knowledge of the causes and consequences of functional and cellular plasticity of the myeloid immune system. High-dimensional characterization of the effects of a Western diet on myeloid immune cell progenitor cells revealed a long-term transcriptional and epigenetic reprogramming of the myeloid cell compartment. The formation of an innate immune memory in myeloid progenitor cells leads to lasting inflammatory priming of monocytes, which may directly contribute to the progression of myeloid cell-associated diseases. In addition, single-cell RNA-seq elucidated unreported cellular heterogeneity of the monocyte and dendritic cell compartment in human peripheral blood. A combination of phenotypic and transcriptional analyzes resulted in a precise categorization of the human DC compartment consisting of pDCs, cDC1, two cDC2 subsets, and a deeply characterized preDC subset. Furthermore, a universal strategy for the integration of cellular atlases was conceptualized and applied to establish a consensus map of the human DC and monocyte cell space. This thesis provides mechanistic insights into the cellular composition of myeloid cells and their functional plasticity, which will form the foundation for further investigations into the dynamic changes of the immune cell compartment during diseases and will be critically relevant for designing effective treatments for a wide variety of pathologies linked to myeloid cells.
Investigating the ability of mammalian Membrane Associated RING-CH proteins to modulate respiratory virus infections
Respiratory viruses generally infect epithelial cells lining the upper and lower airways, however subsets of airway immune cells are also susceptible to infection. Of interest, airway epithelial cells (AEC) and airway macrophages (AM) are both susceptible to influenza virus infection, but only AEC support productive virus replication. Constitutive expression and/or induction of intracellular host proteins to limit or block virus replication is a well-known antiviral mechanism, however few such “restriction factors” have been well characterized and the antiviral activity of many putative restriction factors against respiratory viruses has not been reported. A recent RNA-seq study performed in our laboratory compared gene expression between AEC and AM in the presence or absence of influenza infection, allowing us to identify protein families with differential expression between these two cell types in steady-state and/or following virus infection. We hypothesized that amongst genes differentially expressed between AEC and AM would be those that represent novel restriction factors and these might contribute to the abortive versus productive phenotype observed in AM and AEC, respectively, in regard to influenza replication. One of the protein families identified were the membrane-associated RING-CH (MARCH) family proteins. MARCH proteins are E3 ubiquitin ligases involved in the final step of the ubiquitination process. Amongst their functions, MARCH proteins modulate host immune responses by virtue of their ability to control the turnover of multiple immune molecules, including major histocompatibility complex (MHC) proteins. More recent evidence indicates that they can also modulate virus infection, acting as restriction factors against viruses such as human immunodeficiency virus (HIV)-I, or as proviral factors for viruses such as hepatitis C virus (HCV). Based on these findings, this thesis aimed to investigate the ability of MARCH family proteins to modulate infections caused by influenza and other respiratory viruses. First, we generated stable cell lines with doxycycline (DOX)-inducible expression of several human MARCH proteins (MARCH1/2/3/5/6/7/8/9) and evaluated their ability to modulate either the early or late stages of infections caused by influenza A viruses (IAV), respiratory syncytial virus (RSV) or human metapneumovirus (hMPV). Although none of the MARCH proteins tested affected the early stages of IAV infection (as measured by flow cytometry for newly-synthesized viral proteins), inducible expression of MARCH8 was associated with a significant reduction in titres of infectious IAV released from infected cells. Moreover, MARCH8 expression was also associated with a reduced percentage of RSV-infected cells, consistent with its ability to restrict at an early stage of the RSV replication cycle, although this was not explored further in this thesis. We did not observe any significant differences in the ability of any MARCH protein tested to modulate early or late stages of hMPV infection. Subsequent studies have focussed on understanding MARCH8-mediated restriction of influenza virus infections. Next, we characterized the antiviral activity of MARCH8 against influenza viruses. MARCH8 was shown to mediate antiviral activity against a range of influenza viruses relevant to human health, including A/H1N1, A/H1N1pdm09 and A/H3N2 viruses, as well as against an influenza B virus. We demonstrated that expression of MARCH8, but not the closely related MARCH1, was associated with a reduction in the specific infectivity of virus particles released from IAV-infected cells. Moreover, virus particles released from IAV-infected cells in the presence of MARCH8 exhibited altered protein composition and virion morphology, and MARCH8 was incorporated into the nascent virions. Of interest, MARCH8 expression did not alter cell-surface expression of the viral proteins HA, NA and M2, indicating no major defect in their synthesis and transport during viral replication. Overall, these findings are consistent with MARCH8 acting to block a step late in the virus replication cycle, possibly during virus assembly and/or budding from the cell surface. Studies described in this thesis also attempted to determine features of MARCH8 and IAV relevant to MARCH8-mediated restriction of IAV. We used site-directed mutagenesis and reverse genetics to modify lysine residues in the cytoplasmic tails of the viral hemagglutinin (HA), neuraminidase (NA) and matrix protein (M)2 proteins, either alone or in combination. However, none of these substitutions were sufficient to overcome MARCH8-mediated restriction of IAV. We also modified several lysine residues in the viral M1 protein but again did not identify specific residues to abrogate MARCH8-mediated restriction. Currently, the identity of the particular viral and/or host proteins targeted by MARCH8 which result in inhibition of late stage IAV replication are not known. To study MARCH8 itself, we generated mutants lacking E3 ligase activity and confirmed that this was essential for its anti-IAV activity. Furthermore, given that MARCH8, but not the closely related MARCH1, mediated potent anti-IAV activity against some strains of IAV, we generated cell lines with DOX-inducible expression of MARCH1-MARCH8 chimeric proteins in an effort to determine particular domains of MARCH8 critical to its anti-IAV activity. Chimeric proteins contained one or more domains/regions of MARCH1 substituted into the MARCH8 backbone. While all chimeric proteins retained activity against a common target protein (CD86), all of them also retained ability to restrict IAV replication. Based on these studies, we were unable to identify critical domains of MARCH8 that were essential for its antiviral activity against IAV. Finally, we used a MARCH8 knockout (KO) mouse to evaluate the role of endogenous MARCH8 in modulating IAV infection in vivo. Following intranasal infection, MARCH8 KO mice exhibited enhanced weight loss and viral replication in the lungs at day 5 post- infection, although no major differences in virus titres or virus clearance were observed at later time-points. Moreover, we did not observe major differences in soluble inflammatory mediators or inflammatory cells at day 7 or 10 post-infection. These findings are consistent with a role for endogenous MARCH8 in controlling the early stages of IAV infection in vivo but suggest that it does not have a major impact on virus clearance or the development of adaptive immunity in this model. Of interest, siRNA knockdown of endogenous MARCH8 in a human epithelial cell line also resulted in a modest, but significant, reduction in titres of IAV released from infected cells in vitro. Together, the studies presented in this thesis describe and characterize the antiviral activity of MARCH8 against influenza viruses. In addition, they provide preliminary data to indicate that MARCH8 can also mediate antiviral activity against the pneumovirus RSV, albeit by a distinct mechanism. Overall, these findings contribute to a growing body of evidence that MARCH8 plays an important role in modulating infections caused by a range of different viruses with relevance to human health.
Coxiella burnetii control of the host transcription factors TFEB and TFE3
Coxiella burnetii, the etiological agent of the zoonotic disease Q fever, is an obligate Gram-negative intracellular bacterial pathogen that replicates inside the lysosome-derived CCV (Coxiella-containing vacuole) within mammalian hosts. The CCV maintains the degradative and acidic nature of the host lysosome despite C. burnetii directing the massive expansion of this compartment to accommodate the replicating pathogen. To establish this unique replicative niche, C. burnetii requires the Dot/Icm type IV secretion system (T4SS). This T4SS translocates approximately 150 effectors into the host cell to modulate various cellular processes. To date, the functional role of very few of these effectors have been defined. Given the CCV’s origins it is not surprising that C. burnetii infection increases host autophagy and lysosome biogenesis. To investigate this at the protein level, we employed an elegant SILAC based proteome analysis of human cells infected with C. burnetii. This validated that many proteins involved in these processes are increased in abundance during infection. This prompted us to examine the role of the human transcription factor EB (TFEB) and its close homologue TFE3 during C. burnetii infection. TFEB is a master transcription regulator directly controlling the expression of a network of genes responsible for autophagy and lysosome biogenesis. 3 day’s post-infection with C. burnetii, TFEB/TFE3 is activated as demonstrated by TFEB/TFE3 trafficking from the cytoplasm into the nucleus. The nuclear translocation of TFEB/TFE3 appears to be controlled by C. burnetii as blocking bacterial translation with chloramphenicol leads to TFEB/TFE3 movement back into the cytoplasm. siRNA silencing of tfeb and tfe3 additionally demonstrated their contribution towards the intracellular success of C. burnetii. Interestingly, these host factors did not contribute to the replication of C. burnetii but in the absence of TFEB and TFE3 the CCV did not undergo its typical massive expansion. This research was able to demonstrate that C. burnetii induced activation of TFEB/TFE3 was dependent on the Dot/Icm T4SS thus we hypothesized that an effector(s) of this system may manipulate TFEB/TFE3. An unbiased visual screen was conducted to identify effectors that influence this process and two putative C. burnetii effector proteins, namely CBU1701 and CBU2016, were identified. We demonstrated that ectopic expression of these proteins leads to nuclear localisation of TFEB. Subsequent characterisation of the impact of CBU1701 and CBU2016, demonstrated that they influence the host proteome in similar ways but with surprisingly little impact on TFEB- regulated proteins. These results indicated that nuclear localisation of TFEB in response to CBU1701 and CBU2016 may be uncoupled from activation of this host transcription factor. In addition to characterising the impact of these effector proteins on host cellular function, we set out to understand the role they play in intracellular replication and virulence of C. burnetii. Genetic manipulation of this pathogen is in its infancy and remains technically challenging however this study reports the successful production of multiple mutant strains. Initial characterisation experiments demonstrate that CBU1701 and CBU2016 likely make important contributions to the establishment of the C. burnetii intracellular niche. Overall, the research carried out in this thesis has worked towards elucidating the contribution of TFEB and TFE3 to C. burnetii infection and developing an understanding of the molecular players in this process. Significant tool development and foundational findings reported here will pave the way to a deeper understanding of the interplay between intracellular bacterial pathogens and the host response to infection. Additionally, using C. burnetii effector proteins as a novel biological toolbox may uncover important insights that will impact our understanding of a range of human molecular pathways that impact human health.
Advancing the ferret as an immunological model to study B-cell responses
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.
Investigation of rare actinomycetes for novel antimicrobials
Nocardia are a genus of ubiquitous environmental bacteria belonging to the phylum Actinobacteria. Genomics has revealed that Nocardia species are endowed with extensive and varied arrays of secondary metabolite biosynthetic gene clusters with the potential to produce natural products that have antibiotic properties. Furthermore, the abundance of such gene clusters within the Nocardia rivals that of Streptomyces, the signature genus among the Actinobacteria, owed to the fact that Streptomyces species have yielded many clinically used antibiotics. This project aimed to address the current antibiotic resistance crisis and the shortfall in new compounds within the drug discovery pipeline. A range of natural product discovery techniques were utilised amongst different Actinobacteria with a particular focus on a collection of species within the generally overlooked genus Nocardia. This study had three primary objectives, the first was to use a traditional, high-throughput, empirical screen of 169 pathogenic actinomycetes predominantly from the genus Nocardia. These isolates were screened for antibiotic activity on 19 distinct growth media against a panel of five highly prevalent, multidrug resistant pathogens (Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecium and Acinetobacter baumannii). Secondly, whole genome sequencing and bioinformatic interrogation of 100 Nocardia species was conducted to assess their genetic potential to biosynthesise natural products. This facilitated the selection of a single Nocardia isolate which possessed a non-ribosomal peptide synthetase locus that appeared to be unique amongst other Nocardia species. The locus was also transcriptionally silent. Bioengineering using promoter refactoring was employed to activate expression of this gene cluster, the product of which might have potential as a novel antimicrobial. Thirdly, by utilisation of liquid chromatography-mass spectrometry (LC-MS), bioinformatics and molecular networking, a metabolomic approach was employed to gain a global secondary metabolic footprint of ten predicted “biosynthetically talented" Nocardia species grown on five distinct media types. This project identified: (i) A Nocardia sp. with activity against multidrug resistant Acinetobacter baumannii. (ii) Two Streptomyces isolates (Streptomyces cacaoi and Streptomyces sp.) which exhibited antimicrobial activity against multidrug resistant Escherichia coli and Acinetobacter baumannii respectively. Secondary metabolite extracts from each of these producing isolates were investigated by LC-MS/MS and the resulting spectra was assessed for uniqueness through a dereplication data platform developed specifically for bacterial natural product identification. No hits for previously discovered metabolites were obtained suggesting that the antimicrobials discovered within this project appear to be unique and have potential as new drug leads for today’s ever-decreasing antibiotic discovery pipeline. (iii) Four distinct families of bioactive secondary metabolites that were produced by multiple Nocardia species following LC-MS/MS and molecular network analysis. The identified secondary metabolites were correlated with genome sequence data to identify their probable biosynthetic origin in Nocardia species.
Neuroimmune responses in viral infection
The regulation of the immune responses is important in maintaining good health. Interactions between the nervous and immune systems are increasingly studied and widely appreciated to be influential in orchestrating immune responses. T cells express adrenergic receptors (AR) that enable them to respond to neurotransmitters produced by the sympathetic nervous system (SNS), noradrenaline (NA) and adrenaline, inducing downstream signalling and modulating cell functions, although whether this is stimulatory or inhibitory in T cell antiviral responses is unclear. In this thesis, I examine the effects of SNS in various models of viral infection through chemical sympathectomy and treatment with AR agonists. Modulation of sympathetic signals in systemic infections with LCMV had minor influences on T cell responses but resulted in increased viral loads. Notably, the infection results in a loss of tyrosine hydroxylase (TH) positive sympathetic fibres in the spleen as early as day 3 post infection and is reflected by decreased NA splenic NA. The immune response may play a role with interferon g partially contributing to the depletion. Additionally, this thesis also investigates the capability of long-lasting resident memory T cell (TRM) responses in the highly innervated, immune-privileged cornea. Using a model of herpes infection of the cornea, I showed that T cells are effectively recruited to the cornea with a small heterogenous population able to persist following cessation of immune responses. These cells express CD69 and CD103, canonical markers of tissue residency, to varying degrees. Persistence, but not recruitment, of these cells is dependent on antigen availability at the cornea. These memory cells are capable of responding to secondary encounters with antigen. Moreover, circulating memory cells are also able to infiltrate the immune-privileged cornea more efficiently following infection. Together, these results highlight the important nuances in the regulation of immune responses by the nervous system.