Microbiology & Immunology - Theses
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ItemIdentifying novel compounds to eliminate latent HIV infection( 2019)There are currently an estimated 36.9 million people living with human immunodeficiency virus (HIV) (PLWH) worldwide. In the past few decades, the advent of antiretroviral therapy (ART) has significantly reduced the number of deaths associated with this virus. However, ART is not curative. The persistence of HIV latently infected CD4+ T-cells presents the major barrier towards a cure for HIV. Latently infected T-cells are formed when the virus integrates into the host genome of infected cells without ensuing productive infection. Due to these latently infected cells, viral gene expression and production infection rebounds from the integrated viral DNA if ART is ceased. Thus, ART must currently be taken life-long, posing a tremendous economic burden. The “shock and kill” approach is an extensively studied cure strategy that involves the use of pharmacological agents termed latency reversing agents (LRAs) to reactivate or “shock” the latent virus to express viral RNA and proteins. Following the reactivation of latently infected cells, the production of HIV proteins and viral particles was proposed to result in the elimination of these cells through immune-mediated clearance or cytopathic events. Results from clinical trials that involve a single LRA to reactivate latently infected cells in PLWH have not yielded any significant impact on the HIV DNA reservoir. This can be attributed to a number of different reasons that include the potency of the LRAs to reactivate latency, the failure to elicit an effective immune response and the inhibition of T-cell clearance by cytopathic viral proteins. There is clearly a need for more potent LRAs as well as novel strategies that will result in the clearance of these latently infected cells once reactivated. In this thesis, we investigate several novel pro-apoptotic compounds in isolation as well as in combination with LRAs to clear latently infected cells. We have also developed two new methods in which to study the effects of LRAs and pro-apoptotic drugs on latently infected cells. Dual-fluorescent reporter viruses have proven to be useful tools in studying latent HIV infection in vitro. Here we have modified a dual-fluorescent reporter HIV aiming to improve its functional characteristics in a pre-activation model of HIV latency. The new virus termed, DuoAdvance, contains two fluorescent viral reporters: a latent GFP reporter driven by elongation factor 1-alpha (EF1-alpha) and a productive E2 Crimson reporter driven by the HIV long terminal repeat (LTR) (Chapter 2). Using DuoAdvance, we demonstrate that DuoAdvance can successfully infected Jurkat T-cell lines. In a pre-activation model of HIV latency in primary resting CD4+ T-cells, DuoAdvance infection resulted in little to no latent GFP expression. Subsequent analysis of the GFP negative population of cells revealed DuoAdvance infection can result in the production of latently infected cells carrying latent provirus but the expression of the GFP latency reporter was perturbed. Due to the partial expression of this GFP latent reporter in primary resting T-cells, DuoAdvance is limited to use in dividing T-cell lines and potentially a post-activation model of HIV latency using activated CD4+ T-cells, where better expression of the GFP latency and E2 Crimson reporters were seen. Latency reversing agents can reactivate latent HIV but the effects on decreasing HIV DNA in PLWH has been less encouraging. In this thesis, we examine the effects of different pro-apoptotic drugs combined with different LRAs on decreasing HIV DNA in cultures of CD4+ T-cells from PLWH on ART ex vivo. Here we tested a number of LRAs together with several phosphoinositide-3 kinase (PI3K) inhibitors: IPI-443, IPI-3063 and wortmannin, as well as a B-cell lymphoma-2 (Bcl-2) inhibitor venetoclax as our pro-apoptotic drugs. The LRA romidepsin combined with all pro-apoptotic drugs resulted in synergistic decreases in the levels of integrated HIV DNA in the PLWH CD4+ T-cells ex vivo (Chapter 3). Additionally, several other LRA and pro-apoptotic combinations also decreased integrated HIV DNA in CD4+ T-cells ex vivo. All drugs were able to induce HIV viral transcription. Interestingly, we show that the pro-apoptotic drugs alone also led to an increase in HIV transcription and a decrease in HIV DNA. These data demonstrated the select combinations of pro-apoptotic drugs and LRAs together or pro-apoptotic drugs alone can result in a decrease in HIV integrated DNA in CD4+ T-cells from PLWH on ART ex vivo. However, we were unable to distinguish if there was selective death of the reactivated latently infected cells with minimal impacts on uninfected T-cells also in the cell cultures. In order to explore this, we developed a new approach to detect selective cell death (Chapter 4). This method involves the use of PrimeFlow, a HIV RNA in situ hybridisation method combined with branched-DNA technology, together with a cell death stain and analysis of stained cells using flow cytometry. Using this approach, we were able to demonstrate selective cell death in ACH2 T-cell lines treated with a combination of the PMA LRA, and venetoclax or IPI-443 PI3K inhibitor pro-apoptotic drug in a latently infected T-cell line. However, due to the elaborate staining procedure and large cell loss from the multi-step staining procedure, further investigation is required to move this staining approach into testing these drugs upon inducing the selective death of latently infected CD4+ T-cells from PLWH ex vivo. In summary, we have developed two new methods to investigate the effects of LRAs and/or pro-apoptotic drugs on HIV latency. Although further work is required to optimise these methods for use of the novel DuoAdvance fluorescent reporter virus with primary resting CD4+ T-cells for drug testing, or for use of the novel PrimeFlow assay to study the selective impact of these drugs upon latently infected CD4+ T-cell samples from PLWH ex vivo. Most importantly, our work demonstrates novel combinations of pro-apoptotic drug and LRA combinations that can decrease HIV integrated DNA in cultures of CD4+ T-cells from PLWH on ART ex vivo. This has important therapeutic implications for using these drug combinations to deplete latently infected cells in PLWH on ART and additional studies that investigate these combinations in a clinical setting is warranted. In conclusion, our work demonstrates that latency reversal combined with a drug-based strategy to promote apoptosis can eliminate HIV latently infected CD4+ T-cells from PLWH on ART ex vivo and thus this approach holds important potential to lead to HIV remission off ART in PLWH.
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ItemTranscriptional regulation and co-stimulatory signaling in antiviral T cell immunity( 2018)Special AT-rich binding protein-1 (SATB1) is a global chromatin organizer, promoting or repressing gene transcription in mice and human. In this PhD thesis, SATB1 expression was examined in humans across ages and tissues (Chapter 3). The molecular (Chapter 4) and functional (Chapter 5) role of SATB1 was investigated during anti-viral immunity in mice using an influenza (IAV) infection model. Additionally, the effect of CD27-mediated co-stimulation was studied in the context of HIV-1 infection (Chapter 6). SATB1 has pivotal roles during T cell development and maturation, with lineage fate decision in hematopoietic stem cells and gradual changes in SATB1 expression contributing to T cell development in the thymus in mice. In Chapter 3, SATB1 expression was analyzed across lymphocyte compartments from different human tissues and correlated with PD-1 expression in virus-specific CD8+ T cells. SATB1 expression in pediatric and adult donors showed that SATB1 expression was highest in the human thymus with differential expression levels from DN to DP thymocytes and down-regulation of SATB1 in peripheral T cells. Chapter 3 shows that SATB1 expression in the periphery is not static but follows fine-tuned expression dynamics with downregulation from naïve to antigen-specific CD8+ T cells, likely to be antigen- and tissue-dependent. These data led to the hypothesis that fine-tuned SATB1 expression is necessary for maintaining fate-potential in developing and mature, peripheral T cells. Several molecular mechanisms have been identified for gene regulation by SATB1 with wide-range impacts on the overall chromatin landscape. Previous studies in our laboratory showed that SATB1 mRNA levels are high in naïve, but low in effector CD8+ T cells. The impact of SATB1 in repressing transcriptional programs in naïve CD8+ T cells, prior to its downregulation in effector T cells, was addressed in Chapter 4 of this study. ChIP-Sequencing analysis was performed to decipher genomic binding sites of SATB1 in naïve and effector CD8+ T cells. SATB1 ChIP-Seq data demonstrated that SATB1 binding sites were predominately distal to transcriptional start sites, likely to harbor transcriptional enhancer sites, with reduced SATB1 binding sites in effector over naïve CD8+ T cells. To understand the effects of SATB1 on the transcriptional regulation in naïve and IAV-specific CD8+ T cells, SATB1 imposter mice (SATB1imp/imp) were used in this PhD study. In these mice, Satb1 contains a point mutation in the DNA-binding domain encoding position. SATB1 protein expression in SATB1imp/imp mice persists but is dysfunctional with reduced DNA-binding capability. CD8+ T cells from SATB1imp/imp mice showed up-regulation of certain gene profiles, especially at the naïve stage, such as Pdcd1, Ctla4 and Ccl5, characteristic of activated or exhausted T cells. In Chapter 5, an IAV infection model was used, to examine the effects of dysfunctional SATB1 in IAV-specific CD8+ T cell response generation. CD8+ T cell numbers were consistently reduced in SATB1imp/imp mice with significantly reduced IAV-specific CD8+ T cell numbers in lungs on d10 post-infection. SATB1imp/imp CD8+ T cells exhibited an early overexpression of PD-1 from the naïve stage and reduced polyfunctionality within IAV-specific SATB1imp/imp CD8+ T cells. Using a bone marrow chimera approach, in which mice were reconstituted with a mixture of wildtype and SATB1imp/imp-derived lymphocytes, data showed that reduced T cell numbers and PD-1 overexpression are T cell intrinsic in SATB1imp/imp mice. Immunotherapies, including anti-PD-1, anti-CD27 and histone deacetylase inhibitors, are often used in clinical trials to manipulate activation of T cells. In Chapter 6, we used CD27-mediated stimulation to understand the effect on CD4+ T cells with and without HIV-1 infection. CD27 is a co-stimulatory receptor of the TNF-family, expressed on naïve and central memory T cells. Non-permanent stimulation via CD27 leads to increased primary and memory antiviral CD8+ T cell responses in mice. Here, in humans, CD27-mediated stimulation of CD4+ T cells via its ligand CD70 exhibited profound activation potential in vitro, with high CD4+ T cell proliferation and GzmB production. To examine whether this high activation potential could trigger re-activation of viral transcription in latently infected CD4+ T cells, we re-stimulated CD4+ T cells with conventional α-CD28 or CD27-mediated co-stimulation in an in vitro latency model. Unexpectedly, re-stimulation via CD27 of CD4+ T cells led to reduced viral reactivation compared to α-CD28 stimulation of CD4+ T cells. However, similar transcriptional reactivation levels were obtained when CD4+ T cells isolated from HIV+ individuals on ART were re-stimulated with the two protocols. Strikingly, pre-stimulation of CD4+ T cells prior to in vitro HIV-1 infection showed a trend towards reduced HIV-DNA integration and overall infection. This suggests that CD27-mediated stimulation could lead to activation of antiviral mechanisms that reduces CD4+ T cells HIV-1 infection. Overall, this PhD study provides an in-depth understanding of the transcriptional and co-stimulatory regulations of T cell differentiation in response to viral infections. SATB1’s ability to regulate immune checkpoint molecules, such as PD-1 by its DNA-binding capability in antiviral immunity highlights its significance in future PD-1-related cancer and HIV-1 immunotherapy trials used to reverse T cell exhaustion.
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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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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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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.
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ItemStudies on the role of the membrane proximal ectodomain region (MPER) of HIV-1 gp41 in virus transmission and viral glycoprotein antigenic structure.( 2017)The trimeric HIV-1 envelope glycoprotein (Env) complex, gp120-gp41, is the primary viral structure that elicits neutralizing antibodies. The isolation of broadly neutralizing monoclonal antibodies (bNAbs) from HIV-1 infected individuals has identified 5 major sites of vulnerability in Env, including glycan-dependent epitopes at the gp120 apex, gp120-gp41 interface region, the CD4 binding site and gp41. In addition, the membrane proximal ectodomain region (MPER) is the sole target within gp41 of potent bNAbs such as 2F5, 4E10 and 10E8. The aims of this study were to: 1) examine the function and antigenicity of the MPER in cell-free and cell-to-cell virus transmission; 2) To develop novel immunogens with enhanced expression of bNAb epitopes, including the MPER. Site-directed mutagenesis of the MPER of a macrophage-adapted HIV-1 isolate (AD8) showed that cell-free virus infectivity for U87.CD4.CCR5 cells and peripheral blood mononuclear cells (PBMCs) was sensitive to decreases in hydrophobicity in this region, while efficient spreading infection was observed in cultures initiated with cell-associated virus. These data indicate that the structure and/or functionality of the MPER are distinct in the two modes of virus transmission. The analysis was extended to 2 transmitted-founder (T/F) isolates, SC45 and PRB958 that share 82% amino acid identity in Env. Contrasting effects on cell-free virus infectivity were observed with the W666A mutation in the MPER: blockade of infectivity for SC45, whereas infectivity was enhanced for PRB958. As with AD8, both mutants retained the ability to mediate cell-to-cell spread. The sensitivity of cell-free SC45 virus to the MPER mutation appeared to be associated with a decrease in sensitivity to neutralization by the MPER-directed 10E8 bNAb, when compared to PRB958, which was not inhibited by W666A. These data suggest that the MPER of SC45 is in a relatively occluded conformation. In contrast to cell-free virus infection, the cell-to-cell transmission of both SC45 and PRB958 was resistant to neutralization by 10E8, suggesting that the structural context of the MPER in cell-associated virus precludes its interaction with the 10E8 antibody. A domain swapping approach showed that the variable loops 1 and 4 (V1 and V4) of gp120 conferred the disparate infectivity phenotypes. Overall, the data indicate that the MPER adopts distinct conformations in cell-free and cell-associated virus, being inaccessible to the highly potent MPER-directed bNAb 10E8 in the latter. Previous studies identified 2 mutants that exhibited enhanced sensitivity to neutralization by particular bNAbs: ΔN139INN in V1 enhanced neutralization by bNAbs directed to glycan dependent epitopes in gp120, whereas W596L/K601H/D674E enhanced neutralization by MPER-directed bNAbs. The introduction of ΔN139INN and a combination mutation ΔN139INN/W596L/K601H/D674E (ΔN-LHE) into infectious Env-psuedotyped luciferase reporter viruses and non-infectious HIV-like particles (HIVLP) enhanced the expression of multiple bNAb epitopes in both contexts. ΔN139INN enhanced epitope recognition by bNAbs directed to V1V2 and V3 glycan-dependent epitopes, to a complex epitope involving glycans contributed by gp120 and gp41, and to the CD4 binding site, whereas ΔN-LHE enabled better recognition by a MPER-directed bNAb. It is proposed that these novel epitope-enhancing mutations can be applied to new vaccine candidates to potentially elicit protective bNAb responses.
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ItemBiology and biological impact of HIV-1-specific antibody-dependent cellular cytotoxicity( 2015)Background A safe and effective vaccine has eluded the scientific community for over three decades. With the failure of vaccines based primarily on neutralizing antibody (NAb) and cytotoxic T lymphocytes (CTL), attention has been directed towards novel approaches. The partially successful RV144 vaccine trial focused scientific interest on binding Abs, particularly those that mediate Ab-dependent cellular cytotoxicity (ADCC). Anti-HIV-1 ADCC responses have been associated with slower HIV-1 disease progression in most human studies. Whether such ADCC responses can target HIV-1 envelope protein (Env) from diverse subtypes, however, is largely unknown. It is important to define the breadth of ADCC immunity associated with control of HIV-1. Another unexplored aspect of HIV-1-specific ADCC is the effect of combination antiretroviral therapy (cART) on ADCC Abs. Latent HIV-1 remains unaffected by cART and there is considerable research aimed at achieving a cure for HIV-1 by eliminating the latent reservoir. HIV-1-specific ADCC may assist in eliminating reactivated latently infected cells. An understanding of the effect of cART on ADCC will assist in efforts to enhance immune control of HIV-1 under cART. Furthermore, the biological importance of HIV-1-specific ADCC is strongly suggested by the generation of ADCC-escape HIV-1 variants to linear peptide epitopes. HIV-1 peptide-specific ADCC responses associated with escape from immune recognition represent interesting HIV-1 vaccine antigens. It is therefore important to elucidate the mechanism by which these HIV-1 peptide epitopes are expressed and whether or not they induce ADCC-mediated killing of target cells expressing such peptide-antigens. Methods The breadth of ADCC recognition to 10 HIV-1 gp120 and 8 gp140 proteins from the four major HIV-1 subtypes (A, B, C and E) were analysed in plasma from 11 HIV-1 controllers and 11 HIV-1 progressors, primarily infected with subtype B. Studies were carried out using both an assay that assessed ADCC activity based on the activation of natural killer (NK) cells and the rapid flourometric ADCC (RFADCC) assay that measures ADCC-mediated killing of HIV-1 Env-pulsed target cells. The effect of cART on ADCC responses was analysed in a cohort of 49 cART-naive HIV-1-infected subjects from Thailand (mean baseline CD4 count, 188 cells/μL; mean viral load, 5.4 log10 copies/mL). The subjects were followed for 96 weeks after initiating cART. NK cell activation ADCC, RFADCC and Ab-dependent phagocytosis (ADP) assays were performed using serum samples obtained at baseline and after 96 weeks of cART. To elucidate the mechanism of HIV-1 peptide epitope expression in the whole blood NK activation ADCC assay, we fluorescently tagged ADCC peptide epitopes to identify its target cells. We then assessed ADCC-mediated killing of target cells by the measuring the expression of apoptosis marker Annexin V as well as cell numbers. Results ADCC-mediated NK cell activation was significantly broader (p=0.02) and of higher magnitude (p<0.001) in HIV-1 controllers than in HIV-1 progressors. HIV-1 controllers also showed significantly higher magnitude of ADCC-mediated killing of Env-coated target cells than HIV-1 progressors to both HIV-1 subtype B and the heterologous subtype E gp140 (p=0.001). We found good ADCC reactivity to subtype B and E Envs, less cross-reactivity to subtype A and minimal cross-reactivity to subtype C Envs. We observed 35% reduction in HIV-1 Env-specific ADCC-mediated killing of target cells (p<0.001) after 96 weeks of cART. This was corroborated by a significant reduction in the ability of Env-specific ADCC Abs to activate NK cells (p<0.001). Significantly reduced ADP was also observed after 96 weeks of cART (p=0.018). The fluorescent-tagged ADCC peptide epitopes associate with blood granulocytes. The peptide associated granulocytes become targets for ADCC-mediated killing as shown by enhanced expression of Annexin and reduction in cell numbers. Conclusions Plasma from HIV-1 controllers triggers robust ADCC responses that recognize a broad range of HIV-1 Env. Identifying conserved ADCC epitopes that facilitate this broad ADCC response will assist in designing globally relevant ADCC-based HIV-1 vaccines. Our longitudinal study showing that cART results in significant reductions of HIV-1-specific effector Ab responses. This suggests the need for therapeutic vaccines or other immunomodulatory approaches to improve Ab-mediated control of HIV-1 during cART. Our data suggesting that blood granulocytes express HIV-1 peptide ADCC epitopes and are targeted by NK cells for ADCC-mediated killing provide clearer framework to evaluate these antigens as vaccine candidates. Taken together, the work in this thesis advances our knowledge regarding the importance of HIV-1-specific ADCC immunity and suggests avenues to capitalize on ADCC immunity in the control and prevention of HIV-1 infection.
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ItemThe antibodies involved in the antibody dependent cellular cytotoxicity immune response in HIV( 2012)Background: Antibodies that mediate killing of Human Immunodeficiency Virus (HIV) infected cells by NK cells could be a very useful component of a successful HIV vaccine strategy. Antibody dependent cellular cytotoxicity (ADCC) targets HIV-infected cells by utilising the Fc region of specific antibodies bound to NK cells. However, the role of ADCC responses in preventing HIV-1 remains controversial. Previous studies of ADCC activity have been hampered by difficulties identifying and mapping these responses. An intracellular cytokine staining (ICS) technique developed in our laboratory allows us to detect and map HIV-specific ADCC via detecting activation of NK-cells by ADCC on small volumes of blood. There is an imperative to study the antibodies involved in inducing a HIV-specific ADCC response and use this knowledge to develop effective HIV vaccines. Methods: ADCC responses to overlapping HIV-1 consensus peptide pools were analysed using an ICS assay measuring NK cell activation in 83 anti-retroviral therapy (ART)-naïve HIV-infected subjects followed prospectively for 3 years. We mapped 32 responses to individual consensus HIV subtype B 15mer peptides within the Pol protein. The ADCC-assay was also used to analyse autologous virus-derived peptide epitope responses and compared to consensus derived peptides sequence, across a titration of peptide concentrations, to study escape from ADCC immune response. Isolation of the antibodies responsible for Env and Vpu-specific ADCC responses were purified using affinity chromatography and assessed for their viral inhibition activity, purity and neutralisation activity. We also evaluated bulk antibody production methods using EBV transformation of B cells, Mass Spectrometry of the purified ADCC antibodies and the use of fluorochrome bound peptides to identify and select the ADCC-specific B cells. Results: From the 83 subjects 32 Pol-specific responses were identified. Of these 12 have been mapped to regions of Pol and 2 subjects were identified with ADCC-specific responses to 2 different highly conserved Pol epitopes. Fifty-four subjects recognised Env peptides. From 11 mapped Env responses studied, 6 showed a loss of recognition of autologous virus-derived peptides. This suggested a potential pressure ADCC responses apply on HIV. Purification of Env and Vpu-specific ADCC antibodies proved that ADCC inducing antibodies inhibited viral replication in the Antibody-Dependent Cellular Viral Inhibition (ADCVI) assay and they did not involve neutralising activity. Experiments to determine the most optimal ADCC-specific B cell isolation technique indicated that the use of dual fluorochrome labelled peptides offered the most promising results. Conclusions: Targeting ADCC to more conserved proteins, such as Pol and Vpu proteins may be a more effective ADCC-based vaccine approach. Escape from Env-specific ADCC may, unless broad or directed to rare conserved regions within Env, limit the utility of Env-specific ADCC in controlling or preventing HIV. Development of methods to isolate and generate large amounts of ADCC-specific antibodies will assist in defining the utility of HIV-specific ADCC responses and their role in limiting S/HIV infection. The most effective ADCC epitopes can then be engineered into novel HIV vaccines.