Microbiology & Immunology - Theses
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ItemStrategies for the elicitation of broadly neutralising antibodies to the HIV-1 envelope protein( 2017)An effective prophylactic vaccine for HIV will likely require the elicitation of neutralising antibodies directed towards the Envelope protein (Env) of HIV. In particular, broadly neutralising antibodies (bNAbs) capable of mediating neutralisation against a wide variety of HIV strains would be desirable. bNAbs frequently contain a large degree of affinity maturation, required for the development of their neutralisation breadth, thus the induction of affinity maturation during vaccination may be crucial for the elicitation of bNAbs. This thesis aimed to investigate strategies to enhance bNAb elicitation, in particular, to enhance the affinity maturation of anti-Env antibodies. This was attempted by immune targeting of Env to Clec9A and CR2, which have previously been shown to enhance affinity maturation. Targeting to CR2 was achieved by fusing Env to its ligand, C3d, although this was found to be ineffective at enhancing immunogenicity with the soluble protein constructs assessed. Targeting Clec9A was initially investigated using an anti-Clec9A scFv fused to Env, however when this was found to be unable to bind cell-surface Clec9A, the targeting domain was re-engineered as an anti-Clec9A scFab. While cell-surface Clec9A targeting was achieved successfully, this did not alter the parameters of Env immunogenicity measured. This may have been impacted by the immunodominance of the targeting domains, which future studies will need to address. The conserved epitopes of bNAbs are often poorly exposed, and this contributes to the difficulty in eliciting antibodies against these sites, which are often outcompeted by higher affinity interactions directed towards variable regions of Env. Therefore a novel mutation, ΔN, was investigated for its ability to enhance the exposure of bNAb epitopes in soluble Env constructs. The introduction of the ΔN mutation into SOSIP constructs of the AD8 Env strain enhanced the exposure of the epitopes for multiple bNAb specificities. An immunogenicity study in guinea pigs revealed that AD8 ΔN SOSIP elicited significantly higher titres of antibodies able to block the binding of bNAbs whose epitope exposure was enhanced in this protein. By contrast, ΔN-mediated epitope enhancement and preferential bNAb-like antibody elicitation was not observed with a BG505 strain SOSIP immunogen. Thus, the redirection of the immune response to produce bNAb-like specificities by ΔN appears to correlate with its ability to enhance bNAb epitope exposure in the SOSIP immunogen. The majority of bNAbs are extensively mutated such that most Env strains cannot bind to their precursor antibodies, thus identification of Env immunogens able to bind bNAb precursors is required. A panel of Envs isolated early during infection were screened for interaction with multiple bNAb precursors. This screen identified an Env strain, SC45, able to mediate low binding of the precursors of multiple bNAbs when it is expressed in a membrane-bound form. Expression of soluble SOSIP SC45 abrogates the binding to bNAb precursors, however this protein displays favourable biophysical characteristics desirable in a vaccine immunogen. The introduction of the ΔN mutation into SC45 SOSIP results in a large enhancement in PGT121 epitope exposure, and SC45 SOSIP ΔN is, therefore, a highly promising vaccine candidate.
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ItemElucidating unique HIV-1 subtype C genotypic and phenotypic characteristics to develop novel HIV-1 prognostics( 2015)Human immunodeficiency virus type 1 (HIV-1) causes acquired immunodeficiency syndrome (AIDS) by infecting cells of the immune system. HIV-1 infects target cells via an interaction between its envelope glycoproteins (Env), a cell-surface CD4 receptor and one of two coreceptors, CCR5 or CXCR4. The global spread of HIV-1 has enabled its evolution into a number of distinct subtypes. HIV-1 subtype C (C-HIV) accounts for >50% of HIV-1 infections worldwide and >95% of infections in Southern Africa and Central Asia. While relatively little is known about C-HIV, current evidence suggests that C-HIV pathogenesis is distinct from other HIV-1 subtypes. For example, although the pathogenesis of C-HIV is thought to predominantly involve CCR5-using (R5) viruses, we do not have a firm understanding of how frequently CXCR4-using viruses emerge during progressive C-HIV infection, nor do we completely understand the molecular mechanisms associated with coreceptor switching or cellular tropism. Within this thesis, these major gaps in the literature were addressed. Firstly, the coreceptor usage of a large panel of HIV-1 Envs (n=300) cloned from longitudinally collected plasma samples of 21 antiretroviral (ARV) therapy naïve subjects who progressed from chronic to advanced C-HIV infection was characterised. CXCR4-using C-HIV viruses emerged in only one individual. Subsequent Env mutagenesis studies showed that sequence alterations within the third variable loop (V3) region of Env were critical for determining C-HIV CCR5- or CXCR4-usage. Moreover, by demonstrating a statistically significant linkage between the ability of C-HIV Envs to use CCR5 and the alternative coreceptors CCR3, CCR8 and FRPL1 in vitro, these data suggest that in the absence of coreceptor switching, an altered more flexible use of CCR5 by R5 C-HIV Envs may be selected for during progressive C-HIV infection, which may contribute to C-HIV pathogenicity. Naïve and memory CD4+ T-cell subsets are the major cellular targets for infection by HIV-1. This thesis describes the first analysis of R5 and CXCR4-using C-HIV tropism for naïve and memory CD4+ T-cell subsets, including the recently described stem cell memory CD4+ T-cell (TSCM) subset. The studies described herein show that R5 and CXCR4-using C-HIV viruses preferentially infect memory and naïve CD4+ T-cell subsets, respectively, and that TSCM are susceptible to infection by R5 and CXCR4-using viruses. Mutagenesis and sequence analysis studies established sequence alterations within the V3 region of Env as the major determinants of C-HIV tropism for naïve and memory CD4+ T-cell subsets. Finally, the findings of these studies were used to inform the development of PhenoSeq; a suite of in silico prognostic tests that predict whether HIV-1 uses CCR5 or CXCR4 for entry into cells, based on distinctive sequence mutations within the V3 region of Env. Notably, because the ARV drug maraviroc (MVC) is contraindicated in patients harbouring CXCR4-using HIV-1 strains, a pre-MVC treatment prognostic test is required to determine whether patient’s circulating HIV-1 use CCR5 or CXCR4 for entry into cells. Since the current gold standard prognostic tests are expensive, time consuming and highly specialised, the comparatively rapid, user-friendly and free-to-use PhenoSeq platform (www.burnet.edu.au/phenoseq) has the potential to greatly improve patient access to MVC, in particular for HIV-1 infected individuals residing in resource-constrained regions that are burdened the most by the HIV-1 pandemic. In conclusion, the published outcomes of this thesis have the potential to expedite future research into C-HIV pathogenicity, HIV-1 vaccines and ARV treatments, and may improve patient access to MVC.
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ItemHepatitis C virus envelope glycoprotein E2: isolation and characterisation of a functional core domain( 2010)Hepatitis C virus (HCV) is a major indicator of liver disease and over 200 million chronic infections are estimated worldwide. No vaccine is available and current treatments report limited efficacy. A strong neutralizing antibody response is a key determinant in clearance of acute HCV infection although often appears delayed as broadly neutralizing antibodies do not appear until after chronic infection is established. Recent studies suggest that HCV evades neutralization through the accumulation of immune escape variants within a highly variable, immunodominant sequence of the viral envelope glycoprotein E2. The E1 and E2 envelope glycoproteins are transmembrane proteins that are embedded in the viral membrane and mediate virus attachment and entry into liver cells. The E2 glycoprotein has been shown to interact with the cellular receptors scavenger receptor class B type 1 (SRB1) and CD81 during virus entry and is a major target for neutralizing antibodies. There is currently no high-resolution structure of the HCV E2 glycoprotein to further understand its mechanism of viral entry or immune evasion. However, a soluble E2 ectodomain fragment has been identified that can be efficiently secreted from cells and displays CD81 receptor-binding function. The E2 ectodomain has three discrete variable regions interspersed between conserved CD81-binding motifs: the immunodominant hypervariable region 1 (HVR1), hypervariable region 2 (HVR2) and the intergenotypic variable region (igVR). In this study, simultaneous substitution of these variable regions (VRs) with short, flexible linker motifs within diverse E2 ectodomain sequences was described with the retention of both native folding and CD81 binding function. This indicated that the E2 VRs are excluded from the functional core domain of the glycoprotein. The conserved E2 core domain, lacking all three VRs, was further shown to elicit higher titers of broadly neutralizing antibodies than the unmodified E2 ectodomain suggesting that the VRs occlude conserved neutralization-sensitive epitopes within the underlying core domain. The E2 VRs were demonstrated to modulate CD81 binding in a manner consistent with solvent-exposed structures and illustrated a potential mechanism by which the VRs could mediate immune evasion during acute infection. Therefore, the conserved E2 core domain was proposed to represent a novel and improved antigen for redirecting the immune response towards conserved, neutralization-sensitive epitopes within the E2 glycoprotein. Further biochemical and functional characterization of the E2 core domain is also presented towards optimization of this antigen as viable candidate for future vaccine trials.