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1
Johnson, Kenneth. "The Role of Gilt in the Cross Presentation of the Melanoma Antigen gp100." Thesis, The University of Arizona, 2017. http://hdl.handle.net/10150/623465.
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A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.In this study we examine the utility of using CD8+ T cell hybridomas to measure the ability of bone marrow dendritic cells (BMDCs) to internalize cancer proteins and display them to cytotoxic T cells, a process termed cross‐presentation. We test the ability of a newly generated T cell hybridoma called BUSA14 to detect cross‐presentation of the melanoma antigen gp100. BUSA14 produces a dose‐dependent response to human and mouse gp100 peptides. However, cross‐presentation of gp100 by BMDCs using SK‐MEL‐28 human melanoma cell lysates or direct MHC class I‐restricted presentation by B16 murine melanoma cells was not detected. Both SKMEL‐28 and B16 cells express gp100 protein by immunoblot, and gp100 as a membrane bound protein may be concentrated by cell fractionation techniques. We validated our crosspresentation assay with another T cell hybridoma B3Z to detect cross‐presentation of the model antigen ovalbumin. Lastly, we determined that although BUSA14 expresses the coreceptor CD8, BUSA14 lacks CD3 expression, which likely impairs the ability of this hybridoma to respond to engagement of the T cell receptor and contributes to the inability to detect presentation of native gp100 protein. To resolve these issues, we plan to use primary gp100‐specific T cells from pmel mice expressing the same T cell receptor as the BUSA14 hybridoma to detect presentation of gp100 protein. Ultimately, we plan to evaluate the requirements for cross‐presentation of gp100, including a role for gamma‐interferon‐inducible lysosomal thiol reductase (GILT), a disulfide bond reducing enzyme.
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Grimaud, Eva. "Implications des systèmes LIF/gp130/gp190 et OPG/RANK/RANK-L dans la physiologie ostéoarticulaire." Nantes, 2002. http://www.theses.fr/2002NANT24VS.
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La physiopathologie ostéoarticulaire est un vaste domaine dans lequel interviennent de nombreux facteurs et molécules dont la nature et les rôles ne sont pas encore entièrement élucidés. Ce travail présente l'implication de deux systèmes moléculaires, LIF (Leukemia inhibitory factor)/gp130/gp190 et OPG (ostéoprotégérine)/RANK (receptor activator of nuclear factor kappa b)/RANK-L (RANK-ligand) dans le cartilage en développement et dans des pathologies ostéoarticulaires. La première partie de cette étude a mis en évidence la présence du LIF par immunohistochimie dans les chondrocytes hypertrophiques et les bourgeons conjonctivo-vasculaires sur des coupes de fémurs de rat lors de l'ossification endochondrale. Ces résultats ont également été confirmés chez l'homme. La présence du LIF et son expression ont aussi été mises en évidence par la même technique, ainsi que par RT-PCR, dans une tumeur cartilagineuse de rat, le chondrosarcome de Swarm. Ces résultats suggèrent que la cytokine LIF pourrait être impliquée dans la différenciation des cellules cartilagineuses. .Many factors and molecules whose nature and roles are not yet entirely elucidated intervene in osteoarticular physiopathology. This work presents the implication of two molecular systems, LIF (Leukemia inhibitory factor)/gp130/gp190 and OPG (osteoprotegerin)/RANK(receptor activator of nuclear Factor kappa b)/RANK-L (RANK-ligand) in developmental cartilage and ostearticular pathologies. The first part of this study highlighted the presence of LIF by immunohistochemistry in the hypertrophic chondrocytes and the conjonctivo-vascular buds on sections of rat femur during endochondral ossification. These results were also confirmed at human. The presence of LIF and its expression were also highlighted by the same technique, like RT-PCR, in a cartilaginous tumour of rat, the Swarm rat chondrosarcoma. These results suggest that LIF could be implied in the differentiation of the cartilaginous cells. In a second part, the expression of LIF and its receiving chains gp130 and gp190 was studied during various stages of endochondral ossification in vitro with cellular cell line ATDC5. . .
3
DUPLOMB, LAURENCE. "Contribution a l'etude des recepteurs de la cytokine lif : complexe gp130/gp190 et man 6-p/igfii-r." Paris 7, 2001. http://www.theses.fr/2001PA077078.
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Le leukemia inhibitory factor (lif), cytokine de la famille de l'interleukine-6, est une proteine dotee de nombreuses activites biologiques, agissant sur des types cellulaires varies, sains ou tumoraux. Le lif se fixe avec une basse affinite sur la chaine glycoproteique gp190, dont l'heterodimerisation avec la gp130, chaine commune des recepteurs de la famille de l'il-6, forme un recepteur de haute affinite capable de transduire un signal. Dans un premier temps, notre travail decrit la fixation du lif sur un nouveau recepteur membranaire que nous avons identifie et caracterise comme etant le recepteur au mannose 6-phosphate et a l'insulin-like growth factor ii (man 6-p/igfii-r). Nous avons montre que ce recepteur internalise et degrade le lif, mais est incapable de transduire un signal apres fixation de cette cytokine. La seconde partie du travail met en evidence une interaction, via des residus man 6-p, entre la gp130 et le man 6-p/igfii-r, interaction qui peut entrainer une inhibition des effets biologiques des cytokines de la famille il-6. Cette inhibition ne se faisant ni par modulation des taux membranaires de gp130, ni par induction de proteines regulant de facon negative le signal engendre par la cytokine, nous suggerons qu'il s'agisse d'un encombrement sterique aboutissant a un recepteur non fonctionnel : les sites d'interaction entre la cytokine et son recepteur etant masques par le man 6-p/igfii-r soluble, ou le recrutement de la gp130, par la chaine receptrice specifique de la cytokine, impossible. Cette modulation des effets biologiques du lif par le man 6-p/igfii-r nous a permis de reconsiderer l'action de cette cytokine dans le processus cancereux, au cours duquel on peut observer une perte d'expression membranaire ou une secretion de ce recepteur.
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Spiro, Simon George. "The mechanism of action of iminosugars as antiretrovirals." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:7fc4ae01-bdec-49d0-afec-e0f3e9e8f41d.
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Kong, Leopold. "HIV-1 gp120 : flexibility and glycosylation." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.533859.
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Bodemer, Lucia. "Reinigung des nierenspezifischen Glykoproteins gp400 mittels Immunaffinitätschromatographie." [S.l.] : [s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=960425152.
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Chen, Yuxin. "Characterization of Envelope-Specific Antibody Response Elicited by HIV-1 Vaccines: A Dissertation." eScholarship@UMMS, 2001. http://escholarship.umassmed.edu/gsbs_diss/760.
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Despite 30 years of intensive research，an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function.
First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens.
Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells.
Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies.
Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response.
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Chen, Yuxin. "Characterization of Envelope-Specific Antibody Response Elicited by HIV-1 Vaccines: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/760.
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Despite 30 years of intensive research，an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function.
First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens.
Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells.
Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies.
Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response.
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Farfan, Arribas Diego Jose. "DNA Vaccines Against HIV-1: Augmenting Immunogenicity of gp120." Link to electronic thesis, 2002. http://www.wpi.edu/Pubs/ETD/Available/etd-0107102-160706/.
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Li, Yan. "Processing and secretion of human immunodeficiency virus glycoprotein,gp120." Thesis, University of Ottawa (Canada), 1992. http://hdl.handle.net/10393/10962.
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The natural signal sequence of HIV-1 gp120 contains an unusually long hydrophobic domain and five positively charged amino acids. When the gp120 gene was cloned into a baculovirus expression vector under the control of the baculovirus polyhedrin gene promoter, it exhibited an extremely low level of secretion. However, deletion of the signal sequence resulted in the production of large quantities of a nonglycosylated form of gp120 and fusion of honeybee melittin or murine interleukin 3 signal sequences, which contain only one or no positively charged residues, respectively, resulted in a high level of expression as well as glycosylation and secretion. Four charge-altered signal mutants were generated by oligonucleotide-directed mutagenesis. Positively charged amino acids in the natural signal sequence were substituted with neutral amino acids. The results of these experiments showed that the expression and secretion of gp120 was progressively increased by decreasing the positive charge in a stepwise fashion from + 5 to + 3, + 2, and + 1. However, elimination of all five positive charges (leaving a net negative charge of -1 at the NH 2 terminus) caused accumulation of large amounts of a nonglycosylated form of gp120 but decreased the amounts of glycosylated forms of gp120. These signal peptide mutants clearly demonstrate that the positively charged amino acids in the natural signal sequence of HIV-1 gp120 are key factors determining its poor expression and secretion in insect cells. Analysis of intracellular transport and folding of gp120 further indicates that the highly charged uncleaved signal peptide rather than disulfide bond formation is an important factor limiting transport of gp120 from the rough endoplasmic reticulum (RER) to the Golgi apparatus; its presence affects gp120 folding and slows its rate of transport to the cell surface. The requirement for carbohydrate on HIV gp120 in CD4 binding has been the subject of much debate. There have been conflicting reports regarding the role of gp120 glycans in binding to CD4. An important question is whether the carbohydrate itself plays an important role in this interaction. Nonglycosylated and glycosylated forms of gp120 from HIV-1 and HIV-2 were produced using the baculovirus expression system and their CD4 binding properties were determined. The nonglycosylated forms of gp120 generated by either deletion of the signal sequence or synthesized in the presence of tunicamycin failed to bind to CD4. In contrast, highly mannosylated recombinant gp120 bound well to soluble CD4. Enzymatic removal of carbohydrate chains from glycosylated gp120 by endoglycosidase H (endo H) or by a mixture of endoglycosidase F and N-glycanase (endo FNG) in the presence or absence of SDS had little or no effect on the ability of gp120 to bind CD4. The data indicate that carbohydrate chains per se do not play a significant role in interaction between gp120 and CD4 molecules but that N-linked glycosylation is required for correct protein folding that provides the proper conformation for CD4 binding. Analysis of intracellular folding of gp120, using its ability to bind CD4 as a functional assay for overall conformation, further supports the hypothesis that N-linked glycosylation of HIV gp120 plays an essential role in promoting either the correct folding of the protein or in its stabilization.
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Cohen, Carla J. "Characterization of RNA aptamers that bind to HIV-1 gp120." Thesis, University of Oxford, 2006. http://ora.ox.ac.uk/objects/uuid:e218ac2d-7269-49b2-826e-15ab05b7c1a0.
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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.
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Lo, Wing-sheung James. "Phylogenetic footprinting and modeling of the gp130 family of cytokines." Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B31972019.
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Tung, Wai Na Viola. "Sequence analysis and modelling of the gp130 cytokines and receptors." Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B31972251.
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Tung, Wai Na Viola, and 董維娜. "Sequence analysis and modelling of the gp130 cytokines and receptors." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B31972251.
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Lo, Wing-sheung James, and 羅永裳. "Phylogenetic footprinting and modeling of the gp130 family of cytokines." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B31972019.
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McCormick, Adele. "Enhancement of the immunogenicity of recombinant gp120 of HIV-1." Thesis, University of Sheffield, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366115.
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Gajardo, Johanna. "Etudes sur la gp120 du VIH-1 : pathogénèse et immunogénicité." Montpellier 2, 2007. http://www.theses.fr/2007MON20184.
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Moss, Philippa. "Investigations into the mechanisms underlying HIV-1 gp120-associated neurotoxicity." Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/9488.
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HIV-associated distal sensory polyneuropathy is a frequent (~40% prevalence) complication of HIV infection and treatment. It is characterised by a dying back pattern of axonal degeneration, predominantly of nociceptors usually accompanied by neuropathic pain. The HIV envelope glycoprotein, gp120, has recently been identified as a key mediator of axonal degeneration both in vitro and in vivo. We hypothesised that gp120 interacts, in a chemokine receptor-dependant manner, with primary sensory neurons either directly or indirectly via macrophages and/or Schwann cells. Neurite outgrowth of cultured adult rat dorsal root ganglia (DRG) cells was used to assess direct or indirect neurotoxicity. Gp120 induced concentration-dependent neurite degeneration 24h after exposure, which was not restricted to phenotypic subsets of DRG cells. However, gp120 localisation studies indicated that only a minority (approx. 10%) of neurons internalised gp120 before the onset of neurite degeneration suggesting that the direct toxicity was not a predominant mechanism. Therefore, indirect mechanisms of neurotoxicity were investigated. Application of gp120-conditioned macrophage or Schwann cell media to DRG neuronal cultures revealed that gp120-conditioned media also had the capacity to induce neurite degeneration. Using qPCR it was shown that 4hr exposure to gp120 increased transcription of cytokine–related genes, in cultured Schwann cells and macrophages. These gp120-mediated effects were then explored in vivo. The cytokine expression profiles 4hrs following intradermal gp120 injection in rats were similar to the gene upregulation observed in vitro. These findings highlight the complexity of gp120-mediated mechanisms and indicate that macrophages and Schwann cells may play a key indirect role in the pathogenesis of HIV-associated peripheral neuropathy.
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Bonomelli, Camille. "Antigenic and immunomodulatory properties of HIV-1 gp120 N-linked glycosylation." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:6c003958-e16d-4a70-9e58-8f1c98122376.
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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-1JRCSF 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.
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Meyer, Sonja. "Synthese und Konformationsanalyse von V3-Glycopeptiden des GP120 aus dem HIV." [S.l.] : [s.n.], 1999. http://deposit.ddb.de/cgi-bin/dokserv?idn=963471112.
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Wüstefeld, Torsten. "Molekulare Analyse der IL-6-gp130-abhängigen Signalwege während der Leberregeneration." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=966103084.
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Labell, Rachel. "Synthesis and characterization of galactosyl lipids that bind HIV-1 gp120." Diss., The University of Arizona, 2001. http://hdl.handle.net/10150/289764.
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The HIV-1 virus has a protein, gp120, on its surface that is responsible for the initial recognition between the virus and human cells by binding to the CD4 receptor, which is found on many types of human cells. An alternative receptor, galactosylceramide (GalCer), has also been identified. It binds to HIV-1 gp120 and facilitates the infection of human cells via a CD4 independent mechanism. The goal of this research project was to design, synthesize, and test the effectiveness of galactosyl lipids that bind to gp120. A versatile synthesis was developed and used to synthesize five different GalCer analogs. Professor Scott Saavedra and coworkers used total internal reflection fluorescence microscopy (TIRF) to measure quantitative binding affinities to gp120 at equilibrium for each glycolipid analog. A GalCer analog with octadecyl lipid chains and a tetraethylene glycol spacter group had the highest binding affinity of the analogs tested. Monolayers of lipid mixtures were investigated for phase behavior using epifluorescence microscopy. It was determined that GalCer analogs with saturated tails formed domains in monolayers with DOPC. GalCer analogs were also incorporated into liposomes and were subjected to an HIV-1 inhibition assay in Dr. Ahmad's lab. The GalCer analog liposomes showed similar inhibition as GalCer liposomes.
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Nimal, Sonali. "Fusion of cytokine and gp120 encoding DNA enhances DNA vaccine immunogenicity." Thesis, University of Sheffield, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.606685.
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Shrestha, Jenny. "HIV-1 gp120 Mediated Neuronal Deregulation: Unraveling the Molecular Mechanisms Involved." Diss., Temple University Libraries, 2016. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/402666.
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Molecular Biology and GeneticsPh.D.
The advancement in combinatory antiretroviral therapy (cART) has granted people with HIV-1 an improved lifespan by decreasing the likelihood of AIDS-defining illnesses. People diagnosed early in their infection and undergo cART can keep the virus suppressed and live as long as their HIV-negative peers. As of 2015, more than 50% of people living with HIV in the United States are aged 50 and older. With improved life expectancy, individuals living with long-term HIV infection exhibit many clinical characteristics commonly observed in aging such as: cardiovascular disease, lung disease, certain cancers, HIV-Associated Neurocognitive Disorders (HAND), and liver disease (including hepatitis B and hepatitis C), among others. Regarding neurocognitive disorders, HIV/AIDS patients seem to have learning deficits and working memory impairment such as easy forgetfulness and slowness in action, difficulties in concentration, planning, and multitasking in the condition of having a relatively uneventful and well-controlled clinical course with low HIV viral titers. Neuropsychological studies have disclosed cognitive impairment in a substantial (15–50%) proportion of patients, including learning and working memory deficits which may affect their quality of life, adherence to treatment and ultimately result in increased comorbidity. Studies have described cAMP responsive-element binding (CREB)-1 protein - involved in mitochondrial biogenesis, long-term memory and synaptic plasticity - as a key player in protecting neurons and preventing neurodegeneration. However, loss of CREB protein expression and phosphorylation leads to the development of neurocognitive impairments such as learning deficit and working memory alteration. CREB performs its functions by regulating several genes such, PGC-1 and BDNF being the few (key regulators of mitochondrial bioenergetics, synaptic plasticity and long-term memory, respectively). In here, we have shown that exposure of neuronal cells and animals to HIV-1 gp120 protein decreases expression level of phosphorylated CREB and inhibits its function. Therefore, it will lead to altered neuronal communication and mitochondrial functions. Using pharmacological reagent – rolipram (activates cAMP by inhibiting PDE-4), we were able to reverse the effect of Gp120. Rolipram treatment restored CREB expression and functions altered by gp120. During my graduate years, using in vitro and in vivo studies, I was able to determine the mechanisms used by gp120 leading to the loss of; i – energy metabolism; ii – synaptic plasticity; and iii – long-term memory. We partially determined the relation between gp120, CREB and downstream targets of CREB (PGC-1α and/or BDNF). This approach allowed me to further understand the relation between gp120 and mitochondria as well as between gp120 and neuronal communication. Overall, our study has marked a milestone with regards to understanding the role of gp120 and learning deficiency. Our study mechanistically unravels for the first time the relation between HIV-gp120 protein and development of cognitive disorders such as declarative memory impairment that is commonly observed in HIV-1 patients as well as in aged people. Using an intervention (rolipram) approach, to prevent CREB loss of functions, will help establish new therapeutic strategy (high throughput screening) to mitigate cognitive impairments associated with HIV-1 infection in HIV/AIDS patients.
Temple University--Theses
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Decroly, Etienne. "Contribution à l'étude de la glycoprotéine d'enveloppe (GP160) du virus HIV." Doctoral thesis, Universite Libre de Bruxelles, 1994. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/212679.
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Diop, Amadou Gallo. "Mort neuronale et proteine-enveloppe (gp120) du virus de l'immunodeficience humaine." Limoges, 1988. http://www.theses.fr/1998LIMO306B.
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Xu, Haili, Timothy Radabaugh, Zhenqiang Lu, Michael Galligan, Dean Billheimer, Donata Vercelli, Anne L. Wright, Terrence J. Monks, Marilyn Halonen, and Serrine S. Lau. "Exploration of early-life candidate biomarkers for childhood asthma using antibody arrays." WILEY-BLACKWELL, 2016. http://hdl.handle.net/10150/621762.
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Background: Proteomic approaches identifying biomarkers have been applied to asthma to only a very limited extent. Methods: With an antibody array (RayBiotech, Norcross, GA, USA), the relative intensity and rank differences of 444 proteins were compared in 24 plasma samples obtained at age 3, 11 from children with and 12 without asthma diagnoses at ages 5 and 9. Protein candidates identified by antibody array were quantitated by ELISA in an enlarged sample. Proteins found to differentiate children with and without asthma were also examined for association with known Year 1 asthma risk factors, eczema, and wheeze. Results: In the antibody array, four proteins had rank differences between asthma and non-asthma groups (FDR < 0.1). By ELISA, mean log (+/- s.e.m.) erythropoietin (EPO) level (IU/l) was lower (0.750 +/- 0.048 vs. 0.898 +/- 0.035; p = 0.006) and mean (+/- s.e.m.) soluble GP130 (sGP130) level (ng/ml) was higher in the asthma vs. the non-asthma group (302 +/- 13 vs. 270 +/- 8; p = 0.041). The other 2 array proteins (galactin-3 and eotaxin-3) did not differ by ELISA by asthma. EPO related to the asthma risk factor, first year eczema, whereas sGP130 related to first year wheeze. Conclusions: Through two independent assessments, age 3 plasma levels of EPO and sGP130 were found related to childhood asthma.
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Schockmel, Gerard Alphonse. "Construction of a binding site for HIV-1 GP120 in rat CD4." Thesis, University of Oxford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302857.
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Büchsel, Martin [Verfasser]. "Der Einfluss von gp130 auf die axonale Regeneration im ZNS / Martin Büchsel." Ulm : Universität Ulm. Medizinische Fakultät, 2015. http://d-nb.info/1078957592/34.
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Straatmann, Dirk-Rösken [Verfasser] [Akademischer Betreuer]. "Gp130-Rezeptor-vermittelte Regulation der adulten Neurogenese : In-vivo-Untersuchungen an Mausmutanten." Freiburg : Universität, 2014. http://d-nb.info/1115495631/34.
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Cafferty, William Ben Joseph. "The role of gp130 cytokines in regeneration of injured adult sensory neurones." Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.397613.
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Ueki, Yumi. "Identification of an essential role of gp130 and STAT3 in endogenous neuroprotection." Oklahoma City : [s.n.], 2009.
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LAISNEY, ISABELLE. "Bases moleculaires de la specificite d'anticorps monoclonaux neutralisants anti-gp120 (vih-1)." Paris 7, 1996. http://www.theses.fr/1996PA077084.
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Des changements uniques ou multiples, dans la sequence en acides amines au sein de l'interface du complexe antigene-anticorps ont la capacite d'augmenter l'affinite vers des complexes lies de facon plus etroite ou d'abolir l'interaction completement ou de creer de nouvelles specificites. Notre etude porte sur l'interaction anticorps monoclonal-epitope minimal. Elle repose sur l'utilisation de substitutions, introduites par mutagenese aleatoire pour l'anticorps ou par criblage de banques de peptides aleatoires pour l'antigene. Elle vise a definir et comprendre les bases moleculaires de la specificite d'anticorps monoclonaux anti-gp120. Dans un premier temps, nous avons defini des mimotopes potentiels de l'epitope v3, epitope lineaire, ainsi que du site de liaison de cd4, epitope discontinu, portes par la glycoproteine d'enveloppe du vih-1, la gp120. Bien que l'affinite de l'anticorps mesuree pour les differents peptides mimotopes identifies soit plus faible que celle mesuree avec des petides plus longs ou meme avec la proteine entiere - donnee soulignant l'importance des residus flanquants dans la stabilisation de l'interaction - l'ensemble de ces resultats suggere que les bases moleculaires de la specificite sont portees par de petites sequences lineaires, de six acides amines. Dans un second temps, nous avons etudie plus precisement la structure moleculaire de deux epitopes d'anticorps monoclonaux neutralisants anti-v3. Nos resultats nous ont permis de distinguer, au sein de cet epitope, des residus core, residus critiques pour la liaison de l'anticorps, ainsi que des residus permissifs. Les residus permissifs sont definis comme des acides amines pouvant etre substitues sans supprimer l'interaction de l'epitope avec l'anticorps. Nos resultats suggerent que les mutations tolerees, in vivo, sont localisees uniquement a certains endroits et entrainent une diminution dans l'affinite de liaison de l'anticorps, lorsque celle-ci est elevee (10#-#7 a 10#-#8m). De plus, les profils de permissivite de deux anticorps interagissant avec une region identique different
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O'Donoghue, Robert Joseph James. "The role of directed gp130-mediated signalling in bleomycin-induced murine pulmonary fibrosis." University of Western Australia. School of Medicine and Pharmacology, 2008. http://theses.library.uwa.edu.au/adt-WU2009.0111.
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[Truncated abstract] Fibrosis is a feature of many pulmonary conditions, including idiopathic pulmonary fibrosis (IPF), which is characterised by the accumulation of fibroblasts/myofibroblasts and excessive deposition of collagen. IPF is a disease of unknown aetiology that is unresponsive to current therapy and is typically fatal. The inflammatory cytokine interleukin (IL)-6 is elevated in patients with IPF and recent studies have shown that IL-6-induced signalling is altered in lung fibroblasts from patients with IPF. IL-6 belongs to the gp130 cytokine family, which is a group of ten structurally related cytokines, that all require the membrane bound glycoprotein gp130 to activate intracellular signalling pathways. Gp130 activates intracellular signalling through the Shp2-ERK1/2 and STAT1/3 pathways to mediate cellular activities. This thesis tests the hypothesis that gp130-mediated signalling is dysregulated in the development and progression of pulmonary fibrosis. To address this hypothesis, I assessed the role of gp130-mediated signalling in a mouse model of bleomycin-induced lung fibrosis. This thesis utilised two novel gp130 mutant mice strains with directed and enhanced gp130-mediated Shp2-ERK1/2 (gp130¿STAT/¿STAT) or STAT1/3 (gp130757F/757F) signalling. I observed complete protection from fibrosis in gp130¿STAT/¿STAT mice up to 60 days after bleomycin treatment and profound fibrosis in gp130757F/757F mice compared to wt controls. The enhanced fibrosis observed in gp130757F/757F mice was diminished by monoallelic deletion of STAT3 (gp130757F/757F;STAT3+/-), identifying gp130-STAT3 signalling as a novel promoter of lung fibrosis. ... In addition, IL-6/11 activation of gp130-mediated signalling modulated transforming growth factor (TGF)-ß-induced effects on adult fibroblast proliferation and myofibroblast differentiation. Interaction between IL-6/11 and TGF-ß1 on fibroblast proliferation was dependent on both the gp130-ERK1/2 and gp130-STAT1/3 pathways. Loss of either pathway abrogated the effects of IL-6 and IL-11 on TGF-ß1- 4 induced fibroblast proliferation. However, it was clear that gp130-STAT3 signalling inhibited TGF-ß1-induced myofibroblast differentiation of primary lung fibroblasts. The inhibition of myofibroblast differentiation was associated with gp130-STAT3 dependent inhibition of TGF-ß1-induced Smad3 phosphorylation. These results indicate that IL-6 and IL-11 promote myofibroblastic differentiation of lung fibroblasts, while gp130-STAT3 signalling inhibits TGF-ß1-induced Smad3 phosphorylation and myofibroblastic differentiation of lung fibroblasts While the pathogenesis of IPF is unknown, it is believed that excessive collagen deposition, aberrant fibroblast behaviour and an inflammatory response are critical to the progression of this disease. It has been shown here that IL-6 family cytokines mediate the development and progression of bleomycin-induced lung fibrosis by increasing collagen synthesis, fibroblast proliferation, myofibroblast differentiation and inflammation through gp130-STAT3 signalling. This thesis has demonstrated that differential activation of cytoplasmic signalling pathways by a membrane bound receptor can have a profound effect on pulmonary responses to injury. Furthermore, this thesis is the first study to identify the gp130-STAT3 pathway as a therapeutic target in the treatment of IPF.
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Tjernlund, Annelie. "Leukemia inhibitor factor (LIF) and gp130 in early defence against HIV-1 infection /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7357-039-7/.
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Trillo-Pazos, Gusta. "Toxicity of HIV proteins (NEF, TAT, GP120) and TNFα on human brain cells." Thesis, King's College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341933.
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Taupin, Jean-Luc. "Contribution à l'étude de la cytokine LIF/HILDA et de son récepteur GP190." Bordeaux 2, 1997. http://www.theses.fr/1997BOR28495.
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BAGAYA, BERNARD SSENTALO. "HIV-1 INTERSUBTYPE RECOMBINATION WITHIN GP120 IMPOSES SEVERE FUNCTIONAL RESTRICTION ON RESULTANT ENVELOPES." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1428894939.
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Honke, Nora [Verfasser]. "The regulation of gp130 surface expression on monocytes : implications in chronic inflammation / Nora Honke." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2014. http://d-nb.info/1048606961/34.
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Scanlan, Christopher Neil. "Recognition of a carbohydrate epitope on HIV-1 GP120 : a template for vaccine design." Thesis, University of Oxford, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.427760.
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Yao, Yujing [Verfasser]. "Influence of sialic acid modification on HIV GP120 binding and Syncytia formation / Yujing Yao." Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1176639129/34.
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Kain, Renate. "Identification of gp130 - an antigenic target in small vessel vasculitis expressed on glomerular endothelium." Thesis, University of Aberdeen, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.509165.
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The purpose of my thesis was to capitalise on the development of mammalian expression cloning and the advanced proteomics facilities in Aberdeen to identify gp130. My thesis had four specific objectives: (i) to undertake biochemical experiments to characterise the glycosylation pattern of gp130 and to improve the efficiency of techniques for purifying it both in its native state and after deglycosylation; (ii) to develop a mammalian expression cloning strategy for identifying gp130; (iii) to purify sufficient gp130 for separation on 2-D gels to permit identification of potential candidate molecules by amino acid sequencing using mass spectrometry and Edmann degradation; and (iv) to characterise proteins that co-immunoprecipitate with gp130 and use existing knowledge about these associated proteins to suggest potential candidate molecules for gp130 itself. None of the approaches proved decisive but by combining knowledge gained from all of them I was ultimately able to hypothesise that gp130 was a unique form of aminopeptidase N/CD13 (APN/CD13) and to prove it by showing 293T17 cells transfected with a cDNA encoding CD13 expressed gp130 on their plasma membranes. APN/CD13 in an ectoenzyme otherwise expressed on neo-angiogenic endothelial cells, monocytes and lymphocytes. It has essential roles in angiogenesis and vascular control, and in immunity and inflammation. Future studies will need to determine how Ag11 binding APN/CD13 differs from other forms of CD13 and why this form is selectively expressed by renal microvascular and placental microvascular endothelium. My thesis opens the way for detailed studies of the role of antibodies to APN/CD13 in pauci-immune FNGN.
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Schütt, Antje Michalea [Verfasser]. "Biochemical and physiological analysis of oncogenic constitutive active variants of gp130 / Antje Michalea Schütt." Kiel : Universitätsbibliothek Kiel, 2013. http://d-nb.info/1029710651/34.
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Koshy, Paul John Tharayil. "Cartilage collagen breakdown : the role of interleukin-1 in combination with gp130 binding cytokines." Thesis, University of Newcastle Upon Tyne, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313510.
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Quan, Jun-Min. "Theoretical studies of biomacromolecules : collagen, collagen-like peptides & HIV-1 envelope glycoprotein GP120 /." View abstract or full-text, 2004. http://library.ust.hk/cgi/db/thesis.pl?CHEM%202004%20QUAN.
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Stricher, François. "Développement d'inhibiteurs peptidiques de l'interaction CD4-GP120, exposant des épitopes neutralisants du VIH-1." Paris 11, 2004. http://www.theses.fr/2004PA112067.
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La lutte contre le Virus de l'Immunodéficience Humaine (VIH), confrontée à des problèmes de coût, d'observance et à l'apparition de souches résistantes, a mis en exergue la nécessité de trouver de nouvelles cibles pour inhiber l'infection. L'interaction entre la gp120 et le CD4 est la première étape de l'entrée du virus, donc une cible de choix pour de nouvelles stratégies antirétrovirales. En se basant sur une petite plate-forme peptidique, nous avons développé une mini-protéine reproduisant le site d'interaction du CD4 avec la gp120. Ce peptide, le CD4M33, présente une affinité pour la gp120 proche de celle du CD4, inhibe l'infection par le VIH-1 et est capable d'induire des changements conformationnels similaires à ceux induits par le CD4. Synthétisé chimiquement, il a été couplé à un marqueur fluorescent et nous a permis de développer un test de polarisation de fluorescence spécifique de la liaison CD4-gp120, utile à l'analyse précise de molécules se fixant sur la gp120 au niveau du site d'interaction avec le CD4. Il est adapté au criblage de chimiothèques. Enfin, par une approche de chimie combinatoire, nous avons amélioré le CD4M33 et obtenu des molécules présentant une affinité sub-nanomolaire pour la gp120, des capacités d'inhibition de l'infection accrues et de démasquage des épitopes viraux. Plus que des outils d'analyse de l'interaction CD4-gp120, ces molécules présentent des potentialités thérapeutiques. Elles peuvent être utilisées comme inhibiteurs de l'infection rentrant dans la composition de microbicides, et surtout, comme composants d'un vaccin visant à induire une réponse humorale neutralisante de large spectreFighting the Human Immunodeficiency Virus (HIV)-I is confronted with problems of cost, observance and appearance of resistant strains, thus showing the necessity to find new targets to inhibit the infection. CD4-gp120 interaction being the first step of the virus entry, it represents a perfect target for new antiretroviral strategies. Based on a small peptide scaffold, we engineered a mini-protein reproducing the site of interaction of the CD4 with the gp120. This miniprotein, CD4M33, presents an affinity for gp120 comparable to that of CD4 and inhibits HIV-1 infection of primary cells. Moreover, like CD4, it induces conformational changes in gp120. As it is chemically synthesized, it was coupled to a fluorescent probe and allowed us to develop a fluorescence polarization assay specific for CD4-gp120 binding interaction, useful for analysis of molecules binding gp120 in the CD4 binding site. It is suitable for library screening. Lastly, taking into account the great stability of the scaffold, we further improved CD4M33 by combinatorial chemistry and obtained new peptides presenting sub-nanomolar affinity for gp120, increased capacities to inhibit infection and able to unmask envelope cryptic épitopes of VIH. . More than tools for studying CD4-gp120 binding, these mini-proteins present therapeutic potentialities. They can be used as infection inhibitors, which may enter in the composition of microbicides. Above all, they can be part of a vaccine aiming to induce broadly neutralizing antibodies
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Etter, Jonathan Parker. "Development of Inhibitors in the IL-6/GP130/JAK/STAT Pathway as Therapeutic Agents." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1376525461.
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Magureanu, Camelia-Gabriela. "Autologous and heterologous recognition of monomeric gp120 antigens derived from HIV-1 infected patients." Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/12546.
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The research outlined in this thesis is mainly designed to develop a technology of producing a DNA prime-protein boost vaccine against HIV-1 infection. To reach this aim 1.7kb fragments encoding gp120 antigens deriving from two groups of British HIV-1 infected persons (one consisting of homosexual individuals from Edinburgh, Newcastle and Belfast and the other consisting of haemophiliac patients from Edinburgh who became infected from a common batch of Factor VIII) were PCR amplified and subsequently subcloned into a cloning vector (pGEM T). A mammalian expression vector (pSRHS) was modified in order to include a polylinker to allow the transfer of the 1.7-kb fragments from pGEM T to pSRHS. The recombinant clones were identified and the gp120 genes were expressed in mammalian cells (COS cells) by lipofectin protocol. The functional clones (i.e. those that contained intact open reading frames), were selected and their associated gp120 antigens were quantified by an 'in house' ELISA method. Equivalent amounts of the gp120 antigens were used in an anti-gp120 ELISA to estimate the extent of recognition by the IgG antibodies from autologous and heterologous sera. The nucleic sequences of the functional clones were obtained and some properties such as their predicted NSI/SI phenotype, co-receptor usage and glycosylation sites were analysed. The phylogenetic relationship between the sequences derived from both cohorts was computed and the extent of gp120 antigen recognition by the IgG antibodies from the autologous and heterologous sera was analysed in conjunction with their degree of relatedness. As a conclusion of this study, a high degree of cross-reactivity was noted between antigens and sera, the extent of the recognition of the antigens by the sera was given by the patients' immune status. No significant difference in recognition of the gp120 antigens by sera was observed. This result points towards the potential usage of a cocktail of such DNAs and their corresponding gp120 antigens as a DNA prime-protein boost vaccine.
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Mefford, Megan. "Molecular and Bioinformatic Analysis of Neurotropic HIV Envelope Glycoproteins." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10173.
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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.
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Connell, Bridgette Janine. "Développement d'un test d'interaction entre la protéine d'enveloppe du VIH-1 (gp120) et les corécepteurs CCR5/CXCR4 par résonance plasmonique de surface : criblage et optimisation d'inhibiteurs de l'entrée virale." Thesis, Grenoble, 2012. http://www.theses.fr/2012GRENV013/document.
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Il est bien établi que la gp120 du VIH-1 se fixe aux héparane sulfate (HS) cellulaires, par le biais de la boucle V3 ce qui favorise l'infectivité virale. Cependant, une variété de polyanions solubles, conjugués à CD4 (mCD4-HS12) a des propriétés antivirales et a montré in vitro une activité contre le VIH-1 à de très faibles concentrations (nM). En raison de la complexité structurale des HS, le criblage d'oligosaccharides différenciellement sulfatés pour améliorer l'activité de la molécule serait trop difficile. En vue d'obtenir une molécule plus spécifique, de plus haute affinité et plus facile à produire, des peptides mimant les HS ont été synthétisés par nos collaborateurs. Notre but était de cribler ces peptides pour leur capacité à inhiber l'entrée de VIH-1. À cette fin, nous avons mis en place une plateforme permettant d'immobiliser CCR5 et CXCR4 solubilisés sur des biocapteurs (Biacore) pour cribler des molécules qui inhibent la liaison de gp120-CD4 aux corécepteurs. Pour contrôler le processus de solubilisation, CXCL12, le ligand naturel de CXCR4, a été injecté sur CXCR4 immobilisé. Les affinités des isoformes de CXCL12 (α et γ) pour CXCR4 ont été calculées dans les fourchettes de valeurs précédemment obtenues avec des techniques différentes, prouvant ainsi la fonctionnalité de notre système et nous permettant d'étudier les mécanismes de fixation de ces deux isoformes sur CXCR4 ainsi que leur régulation par HS. Le système a ensuite été utilisé pour cribler la capacité d'inhibition des peptides mimétiques du HS. Chaque peptide, [S(XDXS)n] contient des acides aminés qui imitent les groupes hydroxyles, carboxyles et sulfates des HS. Le peptide contenant des résidus sulphotyrosines, une fois conjugué à mCD4 (mCD4-P3YSO3), montre un IC50 de l'ordre du nM, pour l'inhibition simultanée de la liaison de gp120 aux HS, à CD4, aux anticorps, aux corécepteurs ainsi que l'infection par VIH-1 in cellulo. Il constitue le premier inhibiteur bivalent de l'entrée qui cible à la fois les virus R5 et X4 et le concept d'un peptide mimétique des HS se prête à une analyse structurale et fonctionnelle de la liaison des chaînes HS aux protéines, une nouvelle technique dans ce domainIt is well-established that cell-associated Heparan Sulphate (HS) binds the V3 loop of gp120 of HIV-1 thus aiding in viral infectivity. However, a variety of soluble polyanions have antiviral properties once conjugated to CD4 and a CD4-conjugated HS (mCD4-HS12), showed nM activity against HIV-1 in vitro. Due to the structural complexity of HS, screening differently sulphated oligosaccharides to improve the molecule's activity would be too cumbersome, thus in order to obtain a more specific, higher affinity and easier to produce moiety, collaborators synthesized HS mimetic peptides. We aimed to screen these peptides and other anionic molecules for their capacity to inhibit HIV-1 entry. To this end we set-up a platform whereby solubilised CCR5 and CXCR4 were immobilized on biosensors (biacore) and used to screen for molecules that inhibited gp120-CD4 binding to the coreceptors. To control the solubilization process, CXCL12, the natural ligand of CXCR4, was injected over the immobilized CXCR4. The affinities of CXCL12 isoforms (α and γ) for CXCR4 were calculated within the ranges of values that have been previously described with different techniques, thus proving the functionality of our system and enabling us to investigate the binding mechanisms of these two isoforms with CXCR4 and their regulation by HS. The system was subsequently used to screen the inhibitory capacity of the HS mimetic peptides. Each peptide, [S(XDXS)n], contained amino acids that mimic the hydroxyl, carboxyl and sulphate groups on HS chains. The peptide containing sulphotyrosine residues, when conjugated to mCD4 (mCD4-P3YSO3), displayed nM IC50 for simultaneously inhibiting gp120 binding to HS, CD4, antibody, coreceptors and HIV-1 infection in vitro. This is the first bivalent entry inhibitor that targets both R5 and X4 viruses and the concept of a HSmimetic peptide lends itself to structural-functional analysis of HS chains binding to proteins, a novel technique in this field