Doherty Institute - Theses
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ItemCharacterizing the effects of seminal plasma on anti-HIV immune responses( 2019)Introduction: Semen — an overlooked aspect in models of HIV-1 infection — not only serves as the major vehicle for HIV-1 transmission, but also potently modulates immune responses at the mucosa. Here, we have explored the ability of seminal plasma (SP) to modulate general as well as anti-HIV-1-specific responses by natural killer (NK) cells, cytotoxic T-lymphocytes (CTL), monocytes and neutrophils. These effector cells are potentially important for establishing immunity to HIV-1. Method: The effects of SP on “missing-self” and antibody-dependent responses by NK cells were explored using healthy PBMCs and uncoated 721.221 cells or rituximab-coated 721.221 cells, respectively. In-vitro functional assays were performed in the presence or absence of HIV-1-uninfected and HIV-1-infected SP. Anti-HIV-1-specific NK cell activation and cytolysis were measured by co-incubating healthy PBMCs with gp120-coated CEM.NKr-CCR5 cells in the presence of anti-HIV-1 immune globulin (HIVIg). HIV-1-infected whole blood was stimulated with superantigen Staphylococcus enterotoxin B (SEB) or HIV-1 15-mer Gag peptides to study CTL activation. Redirected cytolysis through CD3 was assessed using healthy PBMCs and P815 target. Activation and cytolysis was assessed utilizing intracellular cytokine staining (ICS) and lactate dehydrogenase (LDH) release assays, respectively. The phagocytic potential and oxidative burst responses of monocytes and granulocytes were measured in healthy whole blood using the PhagoTest and PhagoBurst kits, respectively. The RFADCC assay was used to quantify anti-HIV-1 antibody-mediated responses by monocytes and purified neutrophils through co-incubation with gp120-coated CEM.NKr-CCR5 cells in the presence of HIVIg. The impact of active HIV-1 or bacterial infections on the immunosuppressive capacity of SP was studied using NK cell activation assays. Paired SP samples were collected from HIV-1-infected individuals pre-ART initiation or during-ART, and during the presence or absence of chlamydia and/or gonorrhea infection(s). Finally, through a series of biochemical fractionation and mass spectrometry analyses, we narrowed down the suppressive factor(s) involved in SP-mediated immune inhibition. Results: SP (1:100 dilution) potently suppressed “missing-self” and antibody-mediated activation of NK cells by uncoated 721.221 cells and rituximab-coated 721.221 cells, respectively. Likewise, SP (1:1000 dilution) significantly impaired NK cell-mediated cytolysis against either uncoated 721.221 cells or rituximab-coated 721.221 cells. This inhibition of NK cell activity was observed with both HIV-1-uninfected and HIV-1-infected SP in a dose-dependent manner. Anti-HIV-1 antibody-dependent NK cell activation and cytolysis was potently inhibited by SP (1:100 and 1:1000 dilutions respectively). SP (1:100 dilution) also suppressed CTL activation towards both SEB and HIV-1 15-mer Gag peptide. T-cell mediated cytolysis was also impaired by the presence of SP (1:1000 dilution). In contrast, a higher SP concentration (1:10 dilution) was needed to suppress the phagocytic and oxidative burst responses mediated by granulocytes. This was also true for phagocytic (SP 1:1) and oxidative burst responses (SP 1:10) by monocytes. RFADCC responses by granulocytes and monocytes were also only inhibited by SP 1:1. Active HIV-1 or bacterial infection did not impact the suppression of NK cells by SP. Lastly, we narrowed down SP factor(s) involved in the suppression of NK cells to 15 putative metabolites derived largely from four classes – glycerophosphocholines (GPC), polar metabolites of retinol, polar steroids and polar eicosanoids such as prostaglandins. Conclusion: The presented findings highlight that SP may attenuate vaccine-induced effector responses upon HIV-1 mucosal exposure. This possibility should be considered for HIV-1 vaccine development. Further identification of the factor(s) in SP associated with this potent immunosuppression may help with both understanding susceptibility to HIV-1 infection and identifying novel immunomodulatory agents.
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ItemVariation in killer cell immunoglobulin-like receptors and their human leukocyte antigen class I-encoded ligands impacts natural killer cell education and the control of viral infection( 2018)The interaction of HLA class I (HLA-I) proteins with inhibitory receptors such as those of the Killer cell Immunoglobulin-like Receptor (KIR) family play a key role in effector function acquisition and target cell identification by Natural Killer (NK) cells. However, the extent to which polymorphism in the genes encoding both KIR and their HLA-I encoded ligands impacts the capacity of NK cells to respond to virus-infected or transformed cells with reduced expression of HLA-I proteins is unclear. Analyses of KIR expression on NK cells from a large panel of HLA-I typed healthy donors, found little evidence to suggest that the presence of a HLA-I ligand significantly impacted the frequency of KIR expression. In contrast, the NK cell’s capacity to respond to HLA-I-deficient targets was elevated by the presence of cognate KIR/HLA-I-ligand pairs. Focussed analyses of the interaction between KIR3DL1 and HLA-Bw4 allotypes demonstrated that polymorphisms in both the receptor and ligand impacted the strength of the interaction. Moreover, functional analyses showed that the proportion of KIR3DL1+ve NK cells responding to HLA-I-deficient targets strongly correlated with the strength of the interaction between KIR3DL1 and HLA-Bw4 alloypes, a phenomenon often described as education. Similarly, analyses of KIR3DL1/HLA-Bw4 pairs in a cohort of HIV+ve individuals showed a strong correlation with viral loads, implicating NK cell education in the control of HIV. Analysis of viral loads in HIV-infected HLA-B*57:01+ve individuals revealed that those with KIR3DL1 alleles encoding a valine at position 47 had reduced viral loads relative to individuals that possessed alleles with isoleucine at this position. Binding and reporter cell analyses showed that KIR3DL1*005, a common Ile47 encoding allotype, interacted more strongly with HLA-B*57:01 than other allotypes. Strikingly, functional analyses showed that NK cells expressing Ile47 allotypes were more strongly inhibited by HLA-B*57:01 when expressed at low levels than Val47 variants, suggesting that allotypic variation in KIR impacts the capacity of NK cells to mediate immunosurveillance of HLA-I levels. Finally, although HLA-Bw4 allotypes show extensive conservation across residues that made direct contacts with KIR3DL1, the capacity of allotypes such as HLA-B*57:01 and HLA-A*24:02 to inhibit NK cell activation varied markedly. Since immune synapse formation relies on discerning between structurally similar ectodomains, as may be the case for HLA-Bw4 allotypes, the membrane behaviour of these two HLA-I was compared by fluorescence recovery after photobleaching (FRAP). The data revealed that the mobility of HLA-I was influenced by the cell in which they were expressed, however allotypic differences between the mobility of HLA-B*57:01 and -A*24:02 were observed and, via the analyses of chimeric and mutant HLA-I proteins, was found in part to be dependent on their ectodomains. Taken together, the data suggest that polymorphisms in both KIR and HLA-I impact the strength of their interaction, which in turn influences NK cell education and target cell recognition, particularly with regard to the sensitivity to altered HLA-I expression. In the case of KIR3DL1/HLA-Bw4 interaction, this is manifested in changes in viral replication in HIV-infected individuals and likely has significance in other settings including infection and cancer.
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ItemPathways to HIV latency and reactivation in vitro( 2018)Human immunodeficiency virus (HIV) infection remains a major global health issue. Antiretroviral drugs improve life expectancy and significantly reduce the rate of viral transmission; however, we are far from finding a cure for HIV. The major barrier to finding a cure is the persistence of the replication-competent yet transcriptionally silent latent reservoir. Current latency reversal agents (LRA) lack efficacy to eliminate all the latent proviruses from the reservoir. The response to the same LRAs is varied in latently infected cells ex vivo or in vitro. We hypothesised that HIV could generate different populations of latently infected cells that differ in HIV integration sites and response to reactivation by LRAs. We used a Nef-competent EGFP reporter virus to generate infection and to determine the latently infected cells in chemokine-treated CD4+ T cells in vitro. We first demonstrated that EGFP expression is dependent on viral integration and can be used to determine productively expressed and latently induced infected cells in our culture system. Infection and latency were established in both resting untreated and CCL19-treated CD4+ T cells in vitro. Addition of integrase inhibitor, raltegravir, at time of infection reduced the levels of EGFP expression in both T cell conditions, providing evidence that in our culture system EGFP expression is dependent on viral integration. There was a 4-fold reduction in EGFP expression in the CCL19-treated compared to the matched resting untreated cells. The reduction in the EGFP expression following addition of integrase inhibitor strongly suggested that incubating CD4+ T cells with CCL19 favors viral integration in vitro. We subsequently showed that the addition of IL-7 significantly increases the levels of latency in the chemokine-treated CD4+ T cells. Thus, we clearly showed that both resting and chemokine-treated CD4+ T cells are permissive to direct infection with HIV in vitro. However, the effect of CCL19 in the induction of latency is more pronounced with the addition of IL-7. We further asked whether the establishment of latency affects the response to reactivation by LRAs or T cell receptor (TCR) signalling. We used resting CD4+ T cells to establish infection in the pre-activation pathway and used activated T cells as a model for the establishment of infection in the post-activation pathway. Co-culturing EGFP- cells with allogeneic monocytes alone or in combination with an antibody against CD3 (aCD3); we showed a significant increase in EGFP expression from latently infected cells in the pre-activation latency model. Response to allogeneic monocytes in combination with signals derived from aCD3 significantly correlated with T cell proliferation and there was a minimal spontaneous EGFP expression from latently infected cells in this culture. In contrast, allogeneic monocytes alone or in combination with aCD3 reduced the EGFP expression from latently infected cells in the post-activation latency model. There was no correlation between T cell proliferation and viral expression. The level of spontaneous EGFP expression from latently infected cells was high, and the inhibition of EGFP expression by monocytes was dependent on the direct contact between monocytes and T cells. We further showed that the interaction between T cells and monocytes at time of infection induced spontaneous expression, providing evidence that monocyte-T cell interaction at an early time post infection maintains latency in activated T cells. By direct comparison of pre- and post-activation latency in vitro we, therefore, demonstrated that effective strategies to reverse latency would depend on how latency is established. We further profiled the HIV integration sites in pre- and post-activation latency models and showed a significant enrichment of the sites in genic, exon and intron; in sense direction in the introns of pre-activation latency compared to the post-activation models, suggesting preferential integration of proviral DNA in these locations. By indexing genes with integration sites with gene expression available for these genes in GEO dataset using RNA-Seq analysis, we found a set of genes that are not expressed during activation of T cells in response to TCR stimulation. This observation was found across all T cell subsets in the GEO datasets and suggests there is a common mechanism in T cells that allows for viral entry and integration in non-expressing genes. Our study has clearly shown that how latency is established is a critical factor affecting how latency is maintained or reversed in response to LRAs. Understanding the relationship between chromatin status of the genes that are never expressed during activation of T cells and establishment of infection or latency is of interest for designing strategies to induce the expression from latency or to permanently silence the virus.
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ItemPermanent silencing of HIV transcription using triplex forming oligonucleotides: a novel strategy for an HIV cure( 2018)Combination antiretroviral therapy (cART) for HIV infection has significantly reduced morbidity and mortality, however, treatment is lifelong. The main barrier to a cure for HIV is the persistence of long lived latently infected T-cells. Virus can integrate in the host genome and be transcriptionally silenced however, upon reactivation of transcription virus can re-emerge from these latently infected cells. In individuals on ART, reactivation of virus goes undetected but once ART is stopped, reactivation of virus leads to virus replication and rebound. One strategy to eliminate virus rebound after cessation of ART is to permanently silence HIV transcription. Here we explore an alternative approach to silence HIV transcription in CD4+ T cells using triplex formation oligonucleotides (TFO). We hypothesize that TFOs can bind irreversibly to the integrated provirus in a sequence specific manner with limited off-target effects. We assessed TFO activity against the green fluorescent protein (GFP) and HIV in vitro by using uninfected and latently infected cell lines and determined the effects of gold nanoparticles to enhance nuclear localization.
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ItemThe role of antibody-dependent cellular cytotoxicity in curing latent HIV-1 infection( 2018)As of 2017, 36.9 million people worldwide were living with HIV-1, with approximately 21.7 million people treated with antiretroviral therapy (ART) and only a single individual cured of HIV-1. HIV-1 infection has proven difficult to cure as HIV-1 integrates into the genome of host cells and establishes a latent viral reservoir despite ART, necessitating lifelong therapy for the millions of people living with HIV-1. This continues to place a massive economic burden on the health sector, substantiating the need for an HIV-1 cure to end the HIV-1/AIDS pandemic. One of the most extensively studied cure strategies is the ‘shock and kill’ approach, which aims to reactivate HIV-1 expression from latently infected cells and subsequently eliminate the reactivated cells through immune-mediated mechanisms. We hypothesised that antibody Fc effector functions including antibody-dependent cellular cytotoxicity (ADCC) could be harnessed to eliminate the reactivated viral reservoir. This thesis first examined whether antibodies within HIV-1-infected individuals could recognise and eliminate cells reactivated from latency (Chapter 2). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-dependent natural killer (NK) cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on ACH-2 cells likely resulted in the concealment of CD4-induced (CD4i) epitopes on HIV-1 envelope (Env) that are highly targeted by ADCC antibodies within HIV-1-infected individuals. We found that ex vivo-expanded primary CD4+ T cells from HIV-1-infected individuals were modestly susceptible to ADCC mediated by autologous serum antibodies and effector cells. Importantly, ADCC against these ex vivo-expanded CD4+ T cells could be enhanced following incubation with a small-molecule CD4 mimetic compound that exposes CD4i ADCC epitopes on Env. Since HIV-1-specific ADCC antibodies decline in individuals on long-term ART, this thesis next examined whether latency reversal with the histone deacetylase inhibitor panobinostat could provide sufficient antigenic stimulus to boost ADCC antibodies (Chapter 3). We found that in vivo treatment with panobinostat or a short analytical treatment interruption (ATI) of median 21 days was not sufficient to stimulate an increase in HIV-1-specific ADCC antibodies, despite viral rebound in all individuals who underwent the short ATI. In contrast, a longer ATI of 2 to 12 months robustly boosted HIV-1-specific Fc receptor-binding antibodies and ADCC against HIV-1-infected cells in vitro. These results show that there is a delay between viral recrudescence and the boosting of ADCC antibodies, which has implications for strategies aiming to utilise autologous ADCC responses against latently infected cells. Given that the CD4-bound conformation of Env is predominantly recognised by ADCC antibodies within HIV-1-infected individuals, this thesis next examined whether early-stage infected cells in the process of downregulating CD4 were more susceptible to ADCC compared to late-stage infected cells that have fully downregulated CD4 (Chapter 4). We found that both early- and late-stage infected cells were eliminated by antibodies within plasma from HIV-1-infected individuals, even though there was higher binding of plasma antibodies to the CD4-intermediate early-stage infected cells. Our results show that soluble Env and virions derived from the viral inoculum could sensitise early-stage infected cells to ADCC prior to de novo Env expression, resulting in artefactual in vitro ADCC measurements. Future studies will need to develop improved models to address the potential in vivo role for ADCC against cells with nascent HIV-1-infection. Collectively, our studies highlight a potential role for HIV-1-specific ADCC in curative HIV-1 strategies and in controlling HIV-1 infection. Our results suggest that sufficient expression of Env and the expression of appropriate epitopes will be needed for cells reactivated from latency to be eliminated by autologous ADCC antibodies. Future studies will need to optimise latency reversal in vivo and examine strategies to either boost or administer ADCC antibodies to ensure efficient elimination of the reactivated viral reservoir.
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ItemThe interface of nanotechnology and the human immune system( 2017)Harnessing nanomaterials for the benefit of human health has the potential to improve drug delivery, vaccination and diagnostic imaging. However, a greater understanding of the interaction between nanomaterials and the human immune system is required to improve the clinical translation of nanomedicines. Knowledge of the bio-nano interface has arisen largely from studies in cell lines and rodent models, and our poor understanding of bio-nano interactions in primary human systems remains a key knowledge gap in the development of clinical applications of nanomedicine. This thesis uses novel nano-engineered materials to characterise how material properties influence biological outcomes in primary human samples. By investigating the human bio-nano interface, this research has the potential to improve the rational design of novel nanomedicines. Blood is the first tissue encountered by nanomedicines following intravenous administration – the most common delivery method in the clinic. Therefore, human blood was used as both a source of primary blood cells to examine cell association, targeting and activation, and of plasma for the formation of complex biomolecular coronas. Flow cytometry and confocal microscopy were employed to characterise the role of key physicochemical properties of nanoparticles: charge, reactive surface chemistry, and targeting with antibodies and antibody fragments. A range of nano- engineered particles were developed including caveospheres, hyperbranched polymers (HBP), star polymers and pure PEG particles. Attempts were also made to determine how the biomolecular corona formed in human blood influences the observed bio-nano interactions. Using antibody-capture caveosphere nanoparticles, CD4+ and CD20+ human immune cells could be targeted within mixed cell populations following antibody- functionalisation. Moreover, functionalisation with anti-CCR5 antibodies enabled nanoparticle internalisation into HIV-tropic, non-phagocytic CD4+ T cells, a key hurdle in the delivery of nanoparticle-based anti-HIV therapeutics. Nanoparticle charge defined clear patterns of HBP association with blood cells. These patterns varied for nanoparticles of different material and size, and were not defined by the plasma biomolecular corona that forms in blood. Follow up studies demonstrated cationic, but not anionic or neutral, HBPs activated the myeloid subset of dendritic cells – an important cell target for vaccine applications. The effect of surface chemistry was examined using star polymers. Engineering thiol-reactive pyridyl disulfides onto star polymers directed their association with cancer cell lines, platelets (without activating them) and distinct immune cells subsets. Further studies using preclinical polymer vaccine nanoparticles demonstrated clear differences in blood phagocyte clearance based on brush vs. linear architectures of PEG. Lastly, immunologically stealth particles were functionalised with bispecific antibodies to evaluate cell targeting in the presence of complex biomolecular coronas and the impact of targeting moieties on particle stealth properties. Targeted stealth particles demonstrate potential for the targeted delivery of therapeutics or imaging agents in the presence of plasma coronas, with high specificity and only minimal disruption to particle stealth properties. Phagocytic uptake of PEG particles was dependent on the plasma biomolecular corona. Taken together, these findings further our understanding of the interactions between nano-engineered materials and the human immune system. Ultimately, the development of comprehensive human bio-nano principles will contribute to the rational design of novel nanomedicines.
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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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ItemDevelopment of virus-like particles as immunogens for HIV-1 envelope glycoprotein( 2017)A prophylactic vaccine eliciting broadly neutralising antibody (bNAb) responses against HIV-1 envelope glycoprotein (Env) would be optimal to prevent HIV-1 transmission. Replication incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present virion-associated Env with a native-like structure to the immune system to elicit humoral responses against Env. VLP-associated Env better resembles the viral membrane-bound Env encountered by the immune system during HIV-1 infection than recombinant forms of the glycoprotein. This is likely to be critical for induction of bNAb responses. As Env is highly glycosylated, the expression of VLPs bearing a native N-linked glycosylation profile is also important, especially since many known monoclonal bNAbs incorporate N-linked glycans (N-glycans) into their epitopes. The glycosylation profile of Env is heterogeneous with both populations of typical mammalian N-glycans (complex) and under-processed forms (high-mannose). Furthermore, this profile differs depending on the format of Env used, with virus-associated Env bearing predominantly high-mannose N-glycans whereas recombinant Env is decorated with a greater proportion of complex N-glycans. Here, the viral and expression system factors potentially influencing the differing glycosylation profile were investigated. Recombinant AD8 strain gp120 Env was found to bear a greater proportion of high-mannose N-glycans than when expressed on a viral membrane. The virus-associated Env glycosylation was not influenced by the presence of HIV-1 accessory proteins nor the cell-culture conditions during virus expression. Comparison of the glycosylation profile of recombinant and virus-associated Env using the AD8 and JR-CSF strains, suggested that distinct N-glycan profiles may not be universally conserved for all HIV-1 isolates, although further analysis on a wider range of Env strains is required to confirm this observation. An existing single-plasmid VLP expression vector, based upon DNA T cell vaccine plasmids that were proven safe in human trials, was optimised to maximise Env incorporation and particle budding. The unmodified expression cassette generated VLPs with incomplete protease-mediated cleavage of group specific antigen (Gag) and were irregularly sized. The introduction of alternative mutations that completely removed the reverse transcriptase domain, but preserved most other safety mutations, enabled efficient production of protease-processed, mature-form VLPs (mVLPs). Trimeric Env that presented multiple bNAb epitopes was incorporated into mVLPs, which were capable of viral fusion activity at a level approaching that of wild-type virions. The incorporation of Env into mVLPs was increased by replacing the Env transmembrane and cytoplasmic tail domains with those of influenza haemagglutinin (HA-TMCT). The presentation of bNAb epitopes on the HA-TMCT-modified Env was retained, with the exception of some membrane-proximal epitopes. The mVLP-associated Env was stabilised via the introduction of a trimerisation point mutation and disulfide bonds between Env subunits (SOSIP), which improved the presentation of quaternary bNAb epitopes and diminished the exposure of poorly neutralising antibody sites. Vaccination with mVLPs elicited a broader range of Env-specific antibody isotypes than Env presented on immature VLPs or extracellular vesicles. The mVLPs bearing HA-TMCT-modified Env consistently induced anti-Env antibody responses that mediated modest neutralisation activity. These mVLPs are potentially useful immunogens for eliciting neutralising antibody responses that target native Env epitopes on fully-infectious HIV-1 virions.