Search PubMed⌕ Search

Biomedical subjects

D R Burton

Publications and source records attributed to D R Burton.

At least 19 recordsLinked to original sources
PubMed2006

Copper induces conformational changes in the N-terminal part of cell-surface PrPC.

Prion diseases are caused by misfolding of the cellular prion protein, PrPC. In vitro studies have shown that PrP binds copper via the octarepeat region lying within the unstructured N-terminal segment of the protein, but the significance of copper in PrP metabolism remains unclear. Here, six specific antibodies recognizing different epitope regions of PrP were used to measure the effect of copper on the conformation of the molecule at the cell surface. Binding of an antibody, E149, to an epitope within the octarepeat domain of PrP is halved in the presence of copper, whereas binding of antibodies recognizing epitope motifs C-terminal to residue 90 of PrP remain relatively unaltered under equivalent conditions. These experiments strongly suggest that copper induces localized conformational change within the N-terminal portion of cell-surface PrPC.

Amino Acid Sequence↗
PubMed2004

Improved design of an antigen with enhanced specificity for the broadly HIV-neutralizing antibody b12.

In an attempt to design immunogens that elicit broadly HIV-neutralizing antibodies, we recently engineered monomeric HIV-1 gp120 to bind preferentially b12, a broadly neutralizing antibody to the CD4-binding site (CD4bs) on gp120, by mutating four central residues in the CD4bs to alanine and introducing extra N-glycosylation sites potentially to mask unwanted B-cell epitopes. Despite the favorable antigenicity of this mutant, it harbors two potential caveats that may limit its effectiveness to elicit b12-like antibodies: (i) b12-binding affinity is reduced relative to wild-type gp120 and (ii) binding of some non-neutralizing antibodies to the N-terminal C1 region of gp120 is still observed. Here, we sought to correct these potential limitations. By reverting one of the added N-glycosylation sites on the gp120 core, b12 binding was improved without affecting the epitope-masking properties of the original mutant. Furthermore, truncation of the gp120 N-terminus eliminated binding of the anti-C1 antibodies. Finally, based on the binding profiles of additional non-neutralizing antibodies tested here, further N-glycosylation sites were incorporated to mask their corresponding epitopes. The resulting hyperglycosylated gp120 variants bind b12 and another broadly neutralizing antibody, 2G12, with apparent affinities approaching that of wild-type gp120, but do not bind 21 non- or weakly neutralizing antibodies to seven different epitopes on gp120. These hyperglycosylated variants expand our panel of glycoengineered gp120s that are currently being evaluated for their ability to elicit broadly neutralizing antibodies.

Amino Acid Substitution↗
PubMed2002

Characteristics of immunity induced by viral antigen or conferred by antibody via different administration routes.

The characteristics of the immunity induced by viral antigens or conferred by antiviral antibody via different routes of administration were evaluated comparatively. C57BL/6 mice were immunized via intranasal, intradermal or enteric routes with a live recombinant vaccinia virus expressing the respiratory syncytial virus (RSV) F glycoprotein (F.rVV) or RSV, and then challenged intranasally with RSV. Inhibition of RSV replication was observed in the lungs of all the mice; however, only intranasal immunization hindered virus replication in the nose. Lung inflammation, characterized by infiltration of neutrophils and of mononuclear cells was strongest in the intradermally immunized mice, but was observed in all F.rVV immunized mice to various degrees. Intranasal administration of a potently neutralizing human anti-RSV antibody Fab fragment to infected mice inhibited RSV replication in the nose and, when combined with intraperitoneal administration, protected both the lung and the nose in the absence of deleterious lung pathology. These data suggest that intranasal immunization with F.rVV reduces RSV replication in the respiratory tract, but still induces pathological lung inflammation, even though this is milder than that observed following intradermal immunization. Local neutralizing antibody is indispensable for protection in the nose.

Administration, Cutaneous↗
PubMed2001

Crystallohydrodynamics for solving the hydration problem for multi-domain proteins: open physiological conformations for human IgG.

Hydrodynamic methods provide a route for studying the low-resolution conformation--in terms of time-averaged spatial orientation of the Fab' and Fc domains relative to each other--of the human IgG subclasses, IgG1, IgG2, IgG3 and IgG4 in the environment in which many exist naturally---a solution. Representative modelling strategies are now available using 'shell-bead' or 'shell' modelling of the surface of the molecules with the size-independent programme SOLPRO [J. Garcia de la Torre, S.E. Harding, B. Carrasco, Eur. Biophys. J. 28 (1999) 119-132]. The shell model fits to the equivalent inertial surface ellipsoids of the published crystal structures for the Fab' and Fc domains of IgG are made and an apparent hydration delta(app) of 0.51g/g for Fab' and 0.70 g/g for the glycoprotein Fc are obtained, which yield an average value of (0.59+/-0.07) g/g for the intact antibody (2 Fab'+1 Fc). The relative orientations of these domains for each of the IgG subclasses is then found (using where appropriate a cylindrical hinge) from SOLPRO by modelling the Perrin function, P (i.e. 'frictional ratio due to shape') using this delta(app) and experimentally measured sedimentation coefficients. All the IgG subclasses appear as open, rather than compact structures with the degree of openness IgG3>IgG1>(IgG2, IgG4), with IgG3 and IgG1 non-coplanar. The hingeless mutant IgGMcg, with s degrees (20,w) approximately 6.8 S yields a coplanar structure rather similar to IgG2 and IgG4 and consistent with its crystallographic structure. The extension of this procedure for representing solution conformations of other antibody classes and other multi-domain proteins is indicated.

Crystallography, X-Ray↗
PubMed2001

Antibodies inhibit prion propagation and clear cell cultures of prion infectivity.

Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions. Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc. Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC. Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting.

Animals↗
PubMed2001

Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design.

We present the crystal structure at 2.7 angstrom resolution of the human antibody IgG1 b12. Antibody b12 recognizes the CD4-binding site of human immunodeficiency virus-1 (HIV-1) gp120 and is one of only two known antibodies against gp120 capable of broad and potent neutralization of primary HIV-1 isolates. A key feature of the antibody-combining site is the protruding, finger-like long CDR H3 that can penetrate the recessed CD4-binding site of gp120. A docking model of b12 and gp120 reveals severe structural constraints that explain the extraordinary challenge in eliciting effective neutralizing antibodies similar to b12. The structure, together with mutagenesis studies, provides a rationale for the extensive cross-reactivity of b12 and a valuable framework for the design of HIV-1 vaccines capable of eliciting b12-like activity.

AIDS Vaccines↗
PubMed2001

Immobilized prion protein undergoes spontaneous rearrangement to a conformation having features in common with the infectious form.

It is hypothesized that infectious prions are generated as the cellular form of the prion protein (PrP(C)) undergoes pronounced conformational change under the direction of an infectious PrP(Sc) template. Conversion to the infectious conformer is particularly associated with major structural rearrangement in the central portion of the protein (residues 90-120), which has an extended flexible structure in the PrP(C) isoform. Using a panel of recombinant antibodies reactive with different parts of PrP, we show that equivalent major structural rearrangements occur spontaneously in this region of PrP immobilized on a surface. In contrast, regions more towards the termini of the protein remain relatively unaltered. The rearrangements occur even under conditions where individual PrP molecules should not contact one another. The propensity of specific unstructured regions of PrP to spontaneously undergo large and potentially deleterious conformational changes may have important implications for prion biology.

Animals↗
PubMed2001

Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Adult↗
PubMed2001

Molecular immunologic strategies to identify antigens and b-cell responses unique to multiple sclerosis.

Identification of the causative agent of multiple sclerosis (MS) has long eluded investigators and has become the "Holy Grail" of researchers in the field. The immune response in cerebrospinal fluid of patients with MS, indicated by an increased IgG level and the presence of specific oligoclonal bands after electrophoresis, strongly parallels that found in various infectious diseases of the central nervous system. To understand the nature of B-lymphocyte activation in MS, 4 laboratories studied the antigen-binding regions of antibodies found in MS brain demyelinative plaques and cerebrospinal fluid. Each analysis revealed (1) limited germline expression, results not expected for a random bystander response; (2) features consistent with a specific antigen-targeted process; and (3) the clonal expansion of populations of B lymphocytes in MS. The screening of libraries expressing protein products derived from chronic MS plaque messenger RNA with antibodies purified from plaques, cerebrospinal fluid, or serum of patients with MS has thus far not revealed the antigenic target(s) of the MS antibody response. Because putative MS antigens could be in low abundance, the screening of large libraries of random peptides expressed on phage surfaces might offer an alternative approach to identify peptide sequences recognized by MS antibodies. New sophisticated molecular immunologic techniques described herein should enhance our ability to identify putative antigen(s) targets in MS.

Antibodies, Monoclonal↗
PubMed2001

Molecular profile of a human monoclonal antibody Fab fragment specific for Epstein-Barr virus gp350/220 antigen.

Experimental evidence indicates Epstein Barr virus (EBV) envelope glycoprotein gp350/220 elicits a potent virus neutralizing response in the infected human host that may play an important role in restricting viral pathogenesis. In this study, we report the molecular cloning in combinatorial phage display vectors, of the IgG1 repertoire of an individual naturally infected with EBV, and describe the recovery and characterization of a monoclonal antibody recognizing gp350/220. A detailed understanding of the human antibody response in EBV infection will identify antibodies of potential use in anti-viral prophylaxis and will advance the production of more effective vaccine candidates.

Amino Acid Sequence↗
PubMed2001

Autoantibodies to GPI in rheumatoid arthritis: linkage between an animal model and human disease.

In K/BxN T cell receptor-transgenic mice, spontaneous inflammatory arthritis exhibiting many of the features of human rheumatoid arthritis (RA) is initiated by T cells, but is almost entirely sustained by antibodies to the self-antigen glucose-6-phosphate isomerase (GPI). The relevance of these observations to human disease has been questioned. Here we show that 64% of humans with RA, but not controls, had increased concentrations of anti-GPI immunoglobulin G (IgG) in serum and synovial fluid. In addition, the concentrations of soluble GPI in the sera and synovial fluids of RA patients were also elevated, which led to immune complex formation. Using phage-display methods, we cloned a panel of specific high-affinity human monoclonal anti-GPI IgGs from a patient with RA. These antibodies were highly somatically mutated, which was indicative of an affinity-matured response that was antigen driven. Immunohistochemistry of RA synovium showed high concentrations of GPI on the surface of the synovial lining and on the endothelial cell surface of arterioles; this indicated a mechanism by which antibodies to GPI may precipitate joint disease. The results indicate that the immunological events that lead to the development of autoimmune disease in the K/BxN mouse model may also occur in human RA. This data may be used to develop new strategies for therapeutic intervention.

Amino Acid Sequence↗
PubMed2001

Crystallization and preliminary structure determination of an intact human immunoglobulin, b12: an antibody that broadly neutralizes primary isolates of HIV-1.

An intact human immunoglobulin with a full-length hinge has been crystallized for the first time in a form in which all of the Ig domains are ordered. The IgG1 antibody b12 is one of only three known monoclonal antibodies described that potently neutralize a broad range of HIV-1 primary isolates. It binds to an epitope overlapping the conserved CD4 binding site on the viral surface antigen gp120. Hexagonal crystals corresponding to space group R32 were grown from 0.8 M ammonium sulfate, with unit-cell parameters a = b = 271.3, c = 175.2 A and one molecule per asymmetric unit. The crystals diffract to 2.8 A and a preliminary molecular-replacement solution indicates that all 12 Ig domains of the antibody can be resolved.

Crystallization↗
PubMed2001

Strain-specified relative conformational stability of the scrapie prion protein.

Studies of prion biology and diseases have elucidated several new concepts, but none was more heretical than the proposal that the biological properties that distinguish different prion strains are enciphered in the disease-causing prion protein (PrP(Sc)). To explore this postulate, we examined the properties of PrP(Sc) from eight prion isolates that propagate in Syrian hamster (SHa). Using resistance to protease digestion as a marker for the undenatured protein, we examined the conformational stabilities of these PrP(Sc) molecules. All eight isolates showed sigmoidal patterns of transition from native to denatured PrP(Sc) as a function of increasing guanidine hydrochloride (GdnHCl) concentration. Half-maximal denaturation occurred at a mean value of 1.48 M GdnHCl for the Sc237, HY, SHa(Me7), and MT-C5 isolates, all of which have approximately 75-d incubation periods; a concentration of 1.08 M was found for the DY strain with a approximately 170-d incubation period and approximately 1.25 M for the SHa(RML) and 139H isolates with approximately 180-d incubation periods. A mean value of 1.39 M GdnHCl for the Me7-H strain with a approximately 320-d incubation period was found. Based on these results, the eight prion strains segregated into four distinct groups. Our results support the unorthodox proposal that distinct PrP(Sc) conformers encipher the biological properties of prion strains.

Animals↗
PubMed2001

Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12.

Human monoclonal antibody (MAb) b12 recognizes a conformational epitope that overlaps the CD-4-binding site of the human immunodeficiency virus type 1 (HIV-1) envelope. MAb b12 neutralizes a broad range of HIV-1 primary isolates and protects against primary virus challenge in animal models. We report here the discovery and characterization of B2.1, a peptide that binds specifically to MAb b12. B2.1 was selected from a phage-displayed peptide library by using immunoglobulin G1 b12 as the selecting agent. The peptide is a homodimer whose activity depends on an intact disulfide bridge joining its polypeptide chains. Competition studies with gp120 indicate that B2.1 occupies the b12 antigen-binding site. The affinity of b12 for B2.1 depends on the form in which the peptide is presented; b12 binds best to the homodimer as a recombinant polypeptide fused to the phage coat. Originally, b12 was isolated from a phage-displayed Fab library constructed from the bone marrow of an HIV-1-infected donor. The B2.1 peptide is highly specific for b12 since it selected only phage bearing b12 Fab from this large and diverse antibody library.

Antibodies, Monoclonal↗
PubMed2001

Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro.

A major unknown in human immunodeficiency virus (HIV-1) vaccine design is the efficacy of antibodies in preventing mucosal transmission of R5 viruses. These viruses, which use CCR5 as a coreceptor, appear to have a selective advantage in transmission of HIV-1 in humans. Hence R5 viruses predominate during primary infection and persist throughout the course of disease in most infected people. Vaginal challenge of macaques with chimeric simian/human immunodeficiency viruses (SHIV) is perhaps one of the best available animal models for human HIV-1 infection. Passive transfer studies are widely used to establish the conditions for antibody protection against viral challenge. Here we show that passive intravenous transfer of the human neutralizing monoclonal antibody b12 provides dose-dependent protection to macaques vaginally challenged with the R5 virus SHIV(162P4). Four of four monkeys given 25 mg of b12 per kg of body weight 6 h prior to challenge showed no evidence of viral infection (sterile protection). Two of four monkeys given 5 mg of b12/kg were similarly protected, whereas the other two showed significantly reduced and delayed plasma viremia compared to control animals. In contrast, all four monkeys treated with a dose of 1 mg/kg became infected with viremia levels close to those for control animals. Antibody b12 serum concentrations at the time of virus challenge corresponded to approximately 400 (25 mg/kg), 80 (5 mg/kg), and 16 (1 mg/kg) times the in vitro (90%) neutralization titers. Therefore, complete protection against mucosal challenge with an R5 SHIV required essentially complete neutralization of the infecting virus. This suggests that a vaccine based on antibody alone would need to sustain serum neutralizing antibody titers (90%) of the order of 1:400 to achieve sterile protection but that lower titers, around 1:100, could provide a significant benefit. The significance of such substerilizing neutralizing antibody titers in the context of a potent cellular immune response is an important area for further study.

Administration, Intravaginal↗