RNA aptamers with 2'-fluoro-pyrimidine chemistry were previously selected by in vitro evolution to bind to monomeric HIV-1 gp120 from the R5 strain BaL. A group of 36 novel aptamers were cloned and sequenced from the heterogeneous pool and were tested for their ability to bind to gp120. The diversity of the RNA secondary structure of these, and 27 aptamers isolated previously, was analysed using a bioinformatics approach. This showed that eight aptamers contain a common branched motif, and RNA mutagenesis indicated that this structure is probably required for gp120 binding. Chemically synthesised derivatives of one such aptamer, B40, were designed and tested for binding to gp120. Truncation was found to decrease their binding, but the introduction of point mutations to stabilise the branched conformation and 2'-O-dimethylallyl-modified residues to stabilise helices increased binding to levels greater than that of the parental aptamer. The aptamer epitope on gp120 was mapped by testing aptamer binding to alanine-scanning mutants and deletion mutants of gp120 using a novel plate-based assay. This study showed that the aptamer binding site overlaps with the CCR5 epitope and is confined to four key residues at the base of the V3 loop, one of which is highly conserved. This finding may account for the observation that a number of aptamers were shown previously to neutralise a range of HIV-1 R5 clinical isolates in PBMC cultures. Interestingly however, the aptamer was unable to neutralise HIV-1 pseudovirus in a cell line, which is most likely due to the increased levels of cell-surface CCR5 in cell lines compared to PBMC. Future work should focus on identifying the structure and epitopes of other anti-gp120 aptamers as well as testing neutralisation of HIV-1, HIV-2 and SIV by the B40-derived aptamers. These aptamers can be used as tools to investigate the HIV-1 entry pathway and also have the potential to be developed as anti-HIV-1 microbicides.

The HIV-1 surface glycoprotein, gp120, is made of a rapidly mutating protein core and an extensive carbohydrate shield which are, respectively, encoded by the viral genome and synthesised by the host cell. In contrast to host cell glycoproteins however, gp120 contains a population of unprocessed oligomannose-type glycans that interact with host lectins, promote HIV infection, and alter cell signalling. They also form the basis of the epitopes of several broadly neutralising antibodies isolated against HIV, making them a key feature for immunogen design. The mechanistic basis of how HIV glycans are differentially processed by the host cell was demonstrated on a recombinant gp120 model, suggesting that steric occlusion within the patch of densely packed glycans lead to lack of processing by ER and Golgi α-mannosidases. Furthermore, an elevated level of oligomannose-type glycans was evidenced on gp120 isolated from HIV-1_JRCSF virions produced in PBMCs, compared to recombinant material (respectively ~79% and ~29% of total N-linked glycans), along with a subset of highly processed and sialylated, bi-, tri- and tetra-antennary complex-type glycans, which could be involved in direct interaction with key host cell immune receptors and strongly suppress both antibody and T-cell immune responses. The effect of variation in viral production systems was analysed, with envelope glycoprotein derived from pseudoviral particles produced in HEK 293T cells exhibiting predominantly an oligomannose population (98%), compared to gp120 isolated from a single-plasmid infectious molecular clone (56%). Finally, mutation of one or several glycosylation site(s), known to be required for oligomannose-restricted neutralizing antibodies, was shown to induce a subtle redistribution within the oligomannose series whilst maintaining overall oligomannose levels. The gp120 glycan profile is therefore robust to mutations and also remarkably similar across primary viral isolates from Africa, Asia and Europe and consequently represents an attractive target for vaccine development.

HIV-1, the virus that causes AIDS, is spreading at an alarming rate. Subtype C, which accounts for approximately 50% of infections worldwide, and 98% of infections in Southern Africa, is by far the most prevalent form of the virus. Most molecular and biochemical studies have been performed on HIV-1 subtype B isolates and products, however, and there is a relative scarcity of corresponding data on subtype C. It is therefore of crucial importance to study subtype C HIV-1 strains in order to understand their characteristic pathogenic effects and to develop effective treatment strategies. The aim of research in our laboratory is the development of novel treatment strategies, with particular focus on identifying novel Subtype C Env-binding peptide ligands. This necessitates the development of reagents for use in the discovery and testing of these compounds. In line with this, the aim of this project was the production and characterisation of recombinant Subtype C gp120s generated from a recently compiled HIV-1 virus cohort. To this end, the gp160-coding regions of 20 South African Subtype C HIV-1 strains isolated from AIDS patients presenting at the Johannesburg General hospital in 2005 were amplified by PCR and sequenced. The gp160 amplicons were used to amplify and clone the gp120-encoding regions of these isolates. Two clones, pTriEx- FV3 and pTriEx-FV5, originating from CXCR4- and CCR5-utilising strains respectively, were selected for further use. These clones were cotransfected into insect cells together with a baculoviral DNA backbone in order to generate gp120-expressing baculoviruses by homologous recombination. Recombinant baculoviruses were used to infect Sf9 insect cell cultures for expression of recombinant gp120, which was then purified using a combination of lectin affinity chromatography and ion exchange chromatography. In order to determine the functionality and conformational integrity of the recombinant gp120, the ability of these purified gp120s to bind CD4 and a panel of well-characterised monoclonal antibodies against various epitopes on gp120 (F425 A1g8, 2G12, F425 B4a1, F425 B4e8, 48d, 17b, IgG1 b12, 5F7, 4G10, 9301, ID6, Chessie 13-39.1, 654-30D and 670-30D) was assessed. Gp120 from the CXCR4-using isolate, FV3, appeared to have an intact, functional CD4 binding site as measured by its ability to bind to CD4 and the CD4 binding site antibody 654-30D. It showed low binding to the monoclonal antibody 654- 30D, moderate binding to 2G12, Chessie 13-39.1 and 9301, and high binding to ID6, but did not show binding to any of the other antibodies used in the recognition profile. Gp120 from the CCR5-using isolate, FV5, showed low binding to the monoclonal antibodies F425 B4a1 and Chessie 13-39.1, moderate binding to 2G12, and showed good binding to 9301and ID6. FV5 gp120 could not, however, bind to CD4. This is likely to be related to a D368G substitution, a mutation affecting a critical structural determinant of CD4 binding. The lack of CD4-binding activity of this gp120 highlights the importance of Asp368 for CD4 binding and hints at a region vulnerable for therapeutic targeting. Our results also highlight the challenges of developing broadly therapeutic drugs for HIV-1, as well as the importance of investigating the specific biochemical and pathogenic properties associated with subtype C HIV-1.

The rational design of an HIV-1 vaccine immunogen able to induce potent, cross-reactive, neutralising antibodies remains one of the single greatest challenges in the field of vaccine research today. Roughly a dozen broadly neutralising monoclonal antibodies have been isolated to date, and their epitopes represent important vaccination targets. Interestingly, apart from three that identify over-lapping epitopes in gp41, all of the broadly neutralising monoclonal antibodies target epitopes apparent on different conformations of gp120 (including the epitopes of PG9/PG16). Thus the gp120 monomer remains the most ideal template for immunogen design. Recently, epitopes in the C3-V4 region of gp120 have been shown to be major targets for early strain-specific neutralising antibodies in subtype C infected individuals. Autologous neutralising antibodies identify vulnerable sites on the envelope, and understanding the nature of antigenic “hotspots” on gp120 will help to guide rational vaccine design. This study sought to confirm in four individuals that the C3-V4 epitope was in fact apparent on monomeric gp120, and thereafter to better characterise the nature of viral escape from these antibodies. Using magnetic beads coated with one of 16 different recombinant gp120 proteins it was confirmed that the C3-V4 response was aimed at a monomer-specific epitope in all four cases. In two instances these antibodies were shown to contribute to autologous neutralisation, while in a third the existence of quaternary structure specific antibodies that could not be adsorbed with monomeric gp120 made this link impossible. In the forth instance transfer of the C3-V4 region was shown to expose a normally occluded epitope in the CD4 binding site. This research also provided evidence for other epitopes for autologous neutralising antibodies in C3, overlapping with the CD4 binding site and V5. Lastly, by introducing relevant escape mutations into the parental recombinant gp120s and then comparing the ability of these proteins to adsorb out anti-C3 antibodies, it was shown that while these mutations conferred complete resistance to neutralisation they did not prevent the antibodies from binding to their respective epitopes. The extensive characterisation of C3-related epitopes such as those described in this research should no doubt contribute to the rational design of a gp120 based vaccine immunogen aimed at eliciting broad and potent neutralising antibody responses.

HIV gp120 binds CD4+ cells within plasma membrane lipid rafts inducing a conformational change in gp120 that exposes its V3 loop that binds to a chemokine co-receptor, also within lipid rafts, and initiates fusion. Glycosphingolipids (GSLs) may also be bound by gp120. Lipid rafts, enriched with GSLs and cholesterol, are required for HIV entry and therefore the binding of gp120 to GSL-containing vesicles has been studied. Most of the GSL-structures were within the theoretical raft fraction on a discontinuous sucrose gradient while gp120 binding occurred outside of this fraction where a minority of structures migrated. Gb3 fatty acid content modulated binding. Gp120 bound preferentially to structures depleted of cholesterol and binding was enhanced by treating gp120 with CD4. Two water-soluble mimics of Gb3 inhibited gp120 binding to the different structures. The results demonstrate that the aglycone modulation of GSLs alters their receptor function and that the soluble mimics inhibit binding.

HIV gp120 binds CD4+ cells within plasma membrane lipid rafts inducing a conformational change in gp120 that exposes its V3 loop that binds to a chemokine co-receptor, also within lipid rafts, and initiates fusion. Glycosphingolipids (GSLs) may also be bound by gp120. Lipid rafts, enriched with GSLs and cholesterol, are required for HIV entry and therefore the binding of gp120 to GSL-containing vesicles has been studied. Most of the GSL-structures were within the theoretical raft fraction on a discontinuous sucrose gradient while gp120 binding occurred outside of this fraction where a minority of structures migrated. Gb3 fatty acid content modulated binding. Gp120 bound preferentially to structures depleted of cholesterol and binding was enhanced by treating gp120 with CD4. Two water-soluble mimics of Gb3 inhibited gp120 binding to the different structures. The results demonstrate that the aglycone modulation of GSLs alters their receptor function and that the soluble mimics inhibit binding.

HIV-1 is the causative agent for AIDS and has been a major focus of research for the past two decades. Though there is a combination therapy in place known as the “Highly Active Anti-Retroviral Therapy” (HAART), its usefulness is confounded by the generation of escape mutants, a host of side effects, and its prohibitive cost. The most useful alternative would be the prevention of infection by vaccination. Vaccine research has been focused on the use of recombinant protein sub-units of the virus or combinations thereof to elicit a neutralizing response against the virus. These approaches have mostly resulted in a failure to generate broadly cross reactive neutralizing response against primary strains of the virus. The work reported herein is aimed at designing a rigidified version of gp120/gp120 derivatives and understanding the scope of the various antigenic regions in gp120 in generating a neutralization response. Chapter one discusses some general features of the virus and the immune system. The general nature of AIDS, its spread and its immunological characteristics are also described in this chapter. Chapter two discusses the design and NMR structural analysis of gp120 bridging sheet peptide mimics in methanol and water. The structure of gp120 can be loosely divided into two domains (the outer domain and the inner domain) that are linked together by a discontinuous four stranded antiparallel beta sheet known as the bridging sheet. The bridging sheet is known to overlap with the coreceptor binding site of gp120 and hence is a suitable target for designing virus-entry inhibitors. 17b, a neutralizing antibody isolated from an infected individual, is known to bind to this region of gp120. Our aim in this part of the work was to design a four stranded antiparallel beta sheet, based on the sequence of the bridging sheet, that would contain most of the residues involved in 17b binding. NMR and CD studies confirmed that the peptide was well structured in methanol but the structure was largely lost on addition of aqueous solvent. A small population of the peptide was found to be well-folded in aqueous solution. Chapter three discusses the design and characterization of a gp120-CD4D12 single chain. It is well known that the conformation of gp120 changes upon binding CD4 to expose cryptic epitopes, known as CD4i epitopes. In this work we report the generation of a single chain gp120-CD4 construct that has the cryptic epitopes exposed. The construct bound to 17b, a conformation specific antibody against the bridging sheet of gp120, a cryptic epitope, as well as a non-covalent complex of gp120:CD4D12. There was also very insignificant secondary structural change in gp120 upon complex formation with CD4D12 as observed by CD spectroscopy. Immunological studies with DNA and protein vaccination in guinea-pigs indicated that though 17b like antibodies are generated after immunization, they did not contribute towards the neutralization of primary isolates of the virus. It was also observed that it was the anti-CD4D12 antibodies that were responsible for the neutralization by the sera. These studies indicated towards the inability of the bridging sheet to generate effective neutralization response in case of vaccination with gp120/CD4 complexes. Chapter four discusses the design of a mimic of the gp120/CD4 complex. Since it was seen from our previous work that gp120/CD4 complexes generate a large fraction of antiCD4 antibodies and hence are unsuitable for vaccination purposes, we generated a construct with the minimal binding region of CD4. The small fragment of CD4 spanning from 21st residue to 64th residue was inserted in the V1/V2 loop of gp120. The insertion site was designed based on the region of gp120 closest to this fragment and capable of tolerating insertions. This protein did not bind to 17b as well as gp120/CD4 complex but showed a higher binding compared to full length gp120. Further immunological characterization with this protein revealed that it was not capable of generating neutralizing antibodies against the virus. Chapter five discusses the design and execution of a SPR based solution phase competition experiment to find the solution phase binding constant of CD4 and CD4 analogs to gp120. A major problem during the analysis of binding data obtained by SPR is the accurate determination of Rmax, a parameter needed to obtain an accurate equilibrium dissociation constant. In this chapter we have developed a binary as well as a ternary solution phase SPR based assay to accurately determine a solution phase equilibrium binding constant. The binding constants were determined for gp120 binding to CD4D12 and other CD4 analogs. To confirm the validity of the assay, a control antigen:antibody interaction whose equilibrium dissociation constant has been determined by other methods has been used as a test case. Chapter six discusses the design and characterization of V3 peptides inserted in the loop regions of E. coli Thioredoxin (Trx). Trx has earlier been used to display random peptide libraries between the 33rd and the 34th residue. We have constructed three constructs where the peptide has been inserted between the 33rd and 34th residue, between the 74th and 75th residue and between the 84th and 85th residue. The insertion between 74th and 75th position (74V3Trx) was found to be superior to the other two and would be a suitable alternative for display of a random peptide library. The binding of these constructs to 447-52D, a V3 peptide specific antibody was characterized. These were also characterized immunologically, and 74V3Trx was found to generate weakly neutralizing activity against the MN strain of HIV-1. Competition experiments with 447-52D with these sera indicated that there were antibodies generated that could compete out 447-52D binding to gp120 but not in sufficient concentration to provide broad neutralization. Appendix 1 discusses the rational design of disulfides to stabilize proteins based on the analysis of naturally occurring disulfides. In our attempts to design a rigidified version of gp120 we had designed disulfides in gp120 based on its crystal structure. Many of these were disulfides that would span antiparallel adjacent strands. In order to improve the design principles, we analyzed naturally occurring disulfides that span antiparallel adjacent strands and characterized them in terms of their positional preference in a beta sheet. It was found that these disulfides mostly occur on edge strands and are found exclusively between non-hydrogen bonded registered pairs of adjacent antiparallel strands. Mutagenesis on Thioredoxin was performed to verify our results. It was found that disulfides designed between the non-hydrogen bonded pairs of antiparallel strands could significantly stabilize the protein whereas the ones between hydrogen bonded pairs destabilized the protein.

Endothelial dysfunction and/or injury is pivotal to the development of cardiovascular and inflammatory pathology. Endothelial dysfunction and/or injury has been described in Human Immunodeficiency Virus (HIV) infection. Elaboration of circulating markers of endothelial activation, such as soluble adhesion molecules and procoagulant proteins, occurs in HIV infection. Certain endothelial cells, such as those lining liver sinusoids, human umbilical vein endothelial cells, bone marrow stromal endothelial cells or brain microvascular endothelial cells, have been shown to be variably permissive for HIV infection. Entry of virus into endothelial cells may occur via CD4 antigen or galactosyl-ceramide receptors. Other mechanisms of entry including chemokine receptors have been proposed. Nevertheless, endothelial activation may also occur in HIV infection either by cytokines secreted in response to mononuclear or adventitial cell activation by virus or else by the effects of the secreted HIV-associated proteins, gp 120 (envelope glycoprotein) and Tat (transactivator of viral replication) on endothelium. Enhanced adhesiveness of endothelial cells, endothelial cell proliferation and apoptosis as well as activation of cytokine secretion have all been demonstrated. Synergy between select inflammatory cytokines and viral proteins in inducing endothelial injury has been shown. In HIV infection, dysfunctional or injured endothelial cells potentiate tissue injury, inflammation and remodeling, and accelerate the development of cardiovascular disease.

No description available.

Human immunodeficiency virus (HIV) infection of macrophages in brain and other tissues plays an important role in development of HIV-associated neurological disorders and other aspects of disease pathogenesis. Macrophages express low levels of CD4, and macrophage-tropic HIV strains express envelope glycoproteins (Envs) adapted to overcome this restriction to virus entry by mechanisms that are not well characterized. One mechanism that influences this phenotype is increased exposure of the CD4 or CCR5 binding site, which may increase dissociation of soluble gp120 (sgp120) from Env trimers based on structural models. Little is known about spontaneous sgp120 shedding from primary HIV Envs or its biological significance. In this dissertation, we identify genetic determinants in brain-derived Envs that overcome the restriction imposed by low CD4, examine spontaneous sgp120 shedding by these Envs, and explore the biological significance of these findings. Sequence analysis of the gp120 beta-3 strand of the CCR5-binding site bridging sheet identified D197, which eliminates an N-linked glycosylation site, as a viral determinant associated with brain infection and HIV-associated dementia (HAD), and position 200 as a positively-selected codon in HAD patients. Mutagenesis studies showed that D197 and T/V200 enhance fusion and infection of macrophages and other cells expressing low CD4 by enhancing gp120 binding to CCR5. Sgp120 shedding from primary brain and lymphoid Envs was highly variable within and between patients, representing a spectrum rather than a categorical phenotype. Brain Envs with high sgp120 shedding mediated enhanced fusion and infection with cells expressing low CD4. Furthermore, viruses expressing brain Envs with high sgp120 shedding had an increased capacity to induce lymphocyte activation during PBMC infection, despite similar levels of viral replication. Genetic analysis demonstrated greater entropy and positive selection in Envs with high versus low levels of sgp120 shedding, suggesting that diversifying evolution influences gp120-gp41 association. Finally, we examined V3 loop sequences from dual-tropic brain and lymphoid Envs and found that the frequency of R5X4 HIV-1 is underestimated by most predictive bioinformatic algorithms. Together, these studies provide a better understanding of how neurotropic HIV Envs adapt to target cells expressing low CD4, and possible roles of these viral adaptations in disease pathogenesis.