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ItemStrategies for the elicitation of broadly neutralising antibodies to the HIV-1 envelope proteinKing, Hannah Alexandra Dolby ( 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.
ItemDevelopment of virus-like particles as immunogens for HIV-1 envelope glycoproteinGonelli, Christopher Andrew ( 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.
ItemInvestigating the interactions between dendritic cells, T cells and B cells mediated by targeting Clec9AKato, Yu ( 2016)Dendritic cells (DC) are endowed with an array of receptors that can be exploited for immunotherapy. Targeted delivery of antigen to CD8α+ DCs via Clec9A in vivo induces versatile immune responses, most notably potent thymus-dependent humoral responses even in the absence of adjuvant. However, the basis of the immunogenicity of Clec9A-targeted antigen remains incompletely understood. This thesis describes the complex interactions between CD8α+ DCs and T and B cells mediated by Clec9A to promote and/or regulate immunity. Characterization of CD4+ T cells responding to Clec9A-targeted antigens revealed that they had the phenotype, localization pattern and effector functions consistent with T follicular helper cells (TFH) that provide B cell help. Furthermore, targeting Clec9A primed long-lived memory CD4+ T cells capable of robust secondary TFH responses, even in the absence of adjuvant. Thus, in the steady-state Clec9A-targeted CD8α+ dendritic cells are capable of stimulating CD4+ T cells to promote the development of fully polarized TFH cells. Strikingly, Clec9A was also found to mediate direct interactions between CD8α+ DCs and B cells. B cells were rapidly activated through recognition of native antigen presented on the surface of CD8α+ DCs upon Clec9A-targeted immunization. Direct activation of B cells by CD8α+ DCs was critical for optimal Clec9A-mediated antibody responses as it enabled B cells to effectively acquire help from cognate CD4+ T cells at the T/B borders within the spleen and lymph nodes. Thus, the effective triad of interactions mediated by Clec9A drives potent antibody responses in the steady-state. Unlike TFH and B cells that were potently activated in the steady-state, cross-priming of cytotoxic lymphocytes (CTLs) by Clec9A-targeted antigen required co-administration of adjuvant. In contrast to B cells, Clec9A-mediated primary CTL responses were impaired by the presence of CD4+ T cells. Clec9A-mediated MHC II-restricted presentation favoured the expansion of pre-existing Foxp3+ regulatory T cells (Tregs) in the steady-state, which presumably impaired non-Tregs capacity to activate CD8α+ DCs. Collectively, the data presented in this thesis reveal the versatile capacity of CD8α+ DCs to interact with various cell types to promote immunity/tolerance and reinforces the notion that targeting Clec9A in vivo is a promising strategy to exploit for immunotherapy.