Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 30
Filter
Add more filters










Publication year range
1.
Nitric Oxide ; 78: 72-80, 2018 08 01.
Article in English | MEDLINE | ID: mdl-29859918

ABSTRACT

Nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC), an enzyme that catalyzes the conversion of guanosine-5'-triphosphate (GTP) to cyclic guanosine-3',5'-monophophate (cGMP), transduces many of the physiological effects of the gasotransmitter NO. Upon binding of NO to the prosthetic heme group of sGC, a conformational change occurs, resulting in enzymatic activation and increased production of cGMP. cGMP modulates several downstream cellular and physiological responses, including but not limited to vasodilation. Impairment of this signaling system and altered NO-cGMP homeostasis have been implicated in cardiovascular, pulmonary, renal, gastrointestinal, central nervous system, and hepatic pathologies. sGC stimulators, small molecule drugs that synergistically increase sGC enzyme activity with NO, have shown great potential to treat a variety of diseases via modulation of NO-sGC-cGMP signaling. Here, we give an overview of novel, orally available sGC stimulators that Ironwood Pharmaceuticals is developing. We outline the non-clinical and clinical studies, highlighting pharmacological and pharmacokinetic (PK) profiles, including pharmacodynamic (PD) effects, and efficacy in a variety of disease models.


Subject(s)
Enzyme Activators/therapeutic use , Soluble Guanylyl Cyclase/metabolism , Administration, Oral , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/pharmacokinetics , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Clinical Trials as Topic , Drug Discovery , Enzyme Activation/drug effects , Enzyme Activators/administration & dosage , Enzyme Activators/pharmacokinetics , Enzyme Activators/pharmacology , Fibrosis/drug therapy , Humans , Signal Transduction/drug effects
2.
J Clin Pharmacol ; 46(7): 776-84, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16809803

ABSTRACT

The authors studied a potential drug-drug interaction via findings from in vitro and in vivo studies, to assess whether the in vitro system was predictive of in vivo clinical pharmacokinetic outcomes. An in vitro experiment and a clinical study were performed to assess the potential for interaction. The effect of trospium chloride on human P-glycoprotein-mediated transport of [3H]-digoxin was determined in vitro. A randomized, crossover clinical trial in 40 subjects was performed to evaluate the effect of trospium on the pharmacokinetics of digoxin in vivo. The findings from the studies were then compared. The in vitro findings in this study were corroborated by the clinical study via assessment of inhibition and impact on pharmacokinetic parameters. The in vitro system for assessment of a potential interaction of 2 drugs excreted primarily through the kidney was predictive of the pharmacokinetic outcomes obtained from a clinical setting.


Subject(s)
Cardiotonic Agents/pharmacokinetics , Digoxin/pharmacokinetics , Muscarinic Antagonists/pharmacology , Nortropanes/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Adolescent , Adult , Animals , Benzilates , Cardiotonic Agents/adverse effects , Cross-Over Studies , Digoxin/adverse effects , Drug Evaluation, Preclinical , Drug Interactions , Female , Humans , Ketoconazole/pharmacology , Kidney/drug effects , Kidney/metabolism , LLC-PK1 Cells , Male , Middle Aged , Muscarinic Antagonists/adverse effects , Muscarinic Antagonists/pharmacokinetics , Nortropanes/adverse effects , Nortropanes/pharmacokinetics , Predictive Value of Tests , Reference Values , Swine , Transfection
3.
Sex Transm Infect ; 76(2): 126-30, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10858715

ABSTRACT

BACKGROUND: Although the male condom provides a reliable means of preventing HIV transmission, a broader choice of methods is required particularly in circumstances where the negotiation of condom use is difficult. Development of new products that may be effective as topical vaginal microbicides is the focus of a great deal of research activity currently. The novel agent PRO 2000, a naphthalene sulphonate derivative with in vitro activity against HIV and other sexually transmissible pathogens, is one such compound. We have studied the local and systemic safety and tolerance of a vaginal gel formulation of this agent at two concentrations (0.5% and 4%) over a 2 week period of daily exposure in two cohorts of healthy sexually abstinent women (one in London, UK, and the other in Antwerp, Belgium). METHODS: This was a randomised, placebo controlled, double blind, three arm clinical trial conducted on two sites. Macroscopic evidence of genital epithelial changes was sought using colposcopy and evidence of microscopic inflammation was acquired using high vaginal biopsy from predetermined sites (UK cohort only). Blood levels of PRO 2000 were measured and laboratory safety tests, including coagulation screens, were performed. The impact on vaginal ecology was also assessed. RESULTS: 73 women were enrolled across both sites (36 UK, 37 Belgium); 24, 24, 25 in the 4%, 0.5%, and placebo groups respectively. Of these, 70 completed 2 weeks' exposure to the study gel. Three (all in the 4% group) withdrew owing to adverse events which were possibly or probably gel related. Cervicovaginal abrasion was seen colposcopically in three subjects after 14 days of gel use (two in the 4% group and one in the placebo group). Genital ulceration was not seen during gel use in any of the subjects who completed the study. Histological evaluation of vaginal biopsy samples (36 women only) showed evidence of increased inflammatory signs in one participant of the 4.0% group. One volunteer in the placebo group had moderate inflammation at screening and at follow up. Severe inflammation was not seen among any of the subjects tested. Plasma levels of PRO 2000 and laboratory safety tests showed no evidence of systemic absorption. No impact was seen on normal vaginal ecology in the UK cohort where samples were taken 12 hours after the last gel application. CONCLUSION: In this phase I study PRO 2000 gel was found to be generally well tolerated with promising local and systemic safety profiles. The 0.5% gel was better tolerated than the 4% gel as fewer genital epithelial adverse events were seen in the former. Phase II studies are about to begin in sexually active women.


Subject(s)
Antiviral Agents/adverse effects , HIV Infections/prevention & control , Naphthalenesulfonates/adverse effects , Polymers/adverse effects , Vaginal Diseases/chemically induced , Administration, Intravaginal , Adolescent , Adult , Antiviral Agents/administration & dosage , Belgium , Cohort Studies , Double-Blind Method , Epithelial Cells/pathology , Female , Gels , Humans , Middle Aged , Naphthalenesulfonates/administration & dosage , Patient Satisfaction , Polymers/administration & dosage , Sexual Abstinence , Treatment Outcome , United Kingdom
4.
J Pept Res ; 54(1): 32-42, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10448968

ABSTRACT

Through an integrated study of the reactivity of a monoclonal antibody, 803-15.6, with synthetic peptides and native recombinant HIV-1 envelope glycoprotein gp120, we have obtained structure-functional information on a region of rgp120 not yet elucidated by X-ray crystallography. mAb 803-15.6 binds with high affinity and broad cross-clade specificity to the conserved C-terminal region (amino acids 502-516) of HIV-1 rgp120. Phage display selection from a random peptide library identified the core binding motif as AXXKXRH, homologous to residues 502-508. Using quantitative binding analyses, the affinity of mAb 803-15.6 for native, monomeric recombinant gp120HXB2 (rgp120) was found to be similar to that for the synthetic gp120 peptide (502-516). Circular dichroism studies indicate that the synthetic peptide largely has a random coil conformation in solution. The results therefore suggest that the 803-15.6 epitope is fully accessible on rgp120 and that this region of rgp120 is as flexible as the synthetic peptide. Residues 502-504 are on the edge of a putative gp41 binding site that has been postulated to change conformation on CD4 binding. However, the affinity of mAb 803-15.6 for rgp120 is not affected by binding of CD4 and vice-versa. These results suggest either that the 502-504 region does not change conformation upon CD4 binding, or that recombinant gp120 does not undergo the same changes as occur in the native viral gp120-gp41 oligomer. The detailed characterization of the 803-15.6 epitope may be useful for further study of the role of the C5 region of gp120 in the viral attachment and fusion process.


Subject(s)
Epitopes/chemistry , HIV Envelope Protein gp120/chemistry , HIV-1/chemistry , Amino Acid Sequence , Animals , Binding Sites , CD4 Antigens/metabolism , Circular Dichroism , Conserved Sequence , Epitope Mapping , Epitopes/metabolism , Female , HIV Envelope Protein gp120/metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Structure-Activity Relationship , Surface Plasmon Resonance
5.
J Infect Dis ; 180(1): 203-5, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10353881

ABSTRACT

Vaginal gel formulations containing the naphthalene sulfonate polymer PRO 2000 are being developed as topical microbicides to protect against infection with sexually transmitted disease (STD) pathogens. A mouse model was used to determine whether PRO 2000 could protect against genital herpes in vivo. Animals received a single intravaginal application of 15 microL of a 10% PRO 2000 aqueous solution or a 4.0% or 0.5% PRO 2000 vaginal gel formulation 20 s prior to intravaginal challenge with 4.0 log10 pfu of herpes simplex virus type 2. Treatment with the 4.0% gel provided complete protection against infection; treatment with the 0.5% gel or 10% solution provided 81% and 80% protection, respectively. Furthermore, the 4% gel provided significant protection even when viral challenge was delayed until 60 min after treatment. This is the first report to show that PRO 2000 can protect against infection with an STD pathogen in vivo.


Subject(s)
Herpes Genitalis/prevention & control , Naphthalenesulfonates/therapeutic use , Polymers/therapeutic use , Administration, Topical , Animals , Female , Mice , Solutions/therapeutic use , Vaginal Creams, Foams, and Jellies/therapeutic use
6.
Structure ; 7(2): 131-42, 1999 Feb 15.
Article in English | MEDLINE | ID: mdl-10368281

ABSTRACT

BACKGROUND: The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop. RESULTS: We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II beta turn. CONCLUSIONS: The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.


Subject(s)
HIV Envelope Protein gp120/chemistry , Immunoglobulin Fab Fragments/chemistry , Protein Conformation , Amino Acid Sequence , Binding Sites/immunology , HIV Envelope Protein gp120/immunology , Humans , Hydrogen Bonding , Immunoglobulin Fab Fragments/immunology , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Structure-Activity Relationship , Viral Proteins/chemistry
7.
Mol Immunol ; 34(6): 493-503, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9307065

ABSTRACT

Immune activation is mediated by a specific interaction between the T-cell receptor (TCR) and an antigenic peptide bound to the major histocompatibility complex (MHC). T-cell activation can also be stimulated by superantigens which bind to germline-encoded variable domain sequences of certain TCR beta-chains. We have used a surface plasmon resonance biosensor to characterize the molecular interactions between a class II-restricted alphabeta TCR and its superantigen and MHC/peptide ligands. The extracellular domains of the murine D10 TCR (Valpha2, Vbeta8.2) were expressed in insect cells and secreted as a disulfide-linked heterodimer. In the absence of MHC class II, purified soluble D10 TCR bound to Staphylococcus aureus enterotoxin C2 with an association rate of 1.69+/-0.12 x 10(4)M(-1) sec(-1) and a dissociation rate of 1.9+/-0.47 x 10(-2) sec(-1), giving a dissociation constant of 1.1 microM. Binding of the TCR to S. aureus enterotoxin B was barely detectable and could not be measured accurately due to the rapid dissociation rate. Soluble D10 TCR also bound to a soluble murine MHC class II I-A(k) molecule containing a fused antigenic conalbumin peptide and complementary leucine zipper sequences to facilitate efficient chain pairing. The purified I A(k) chimera specifically stimulated proliferation of the D10 T-cell clone, and bound to immobilized soluble D10 TCR with an association rate of 1.07+/-0.19 x 10(4)M(-1)sec(-1) and a dissociation rate of 2.2+/-0.65 x 10(-2) sec(-1), giving a dissociation constant of 2.1 microM.


Subject(s)
Histocompatibility Antigens Class II/metabolism , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Superantigens/metabolism , Animals , Baculoviridae , Biosensing Techniques , Cells, Cultured , Kinetics , Ligands , Lymphocyte Activation , Moths , Peptides/immunology , Peptides/metabolism , Protein Binding , Recombinant Fusion Proteins , Solubility , Spectrum Analysis
8.
J Biol Chem ; 272(51): 32190-7, 1997 Dec 19.
Article in English | MEDLINE | ID: mdl-9405420

ABSTRACT

We recently showed that a soluble, heterodimeric murine D10 T-cell receptor (TCR) (Valpha2Calpha, Vbeta8.2Cbeta) expressed in insect cells binds both Vbeta8.2-specific bacterial superantigen staphylococcal enterotoxin C2 (SEC2) and a soluble, heterodimeric major histocompatibility complex class II I-Ak.conalbumin peptide complex with a low micromolar affinity. To define further the structural requirements for the TCR/ligand interactions, we have produced in Escherichia coli a soluble, functional D10 single chain (sc) TCR molecule in which the Valpha and Vbeta domains are connected by a flexible peptide linker. Purified and refolded D10 scTCR bound to SEC2 and murine major histocompatibility complex class II I-Ak.conalbumin peptide complex with thermodynamic and kinetic binding constants similar to those measured for the baculovirus-derived heterodimeric D10 TCR suggesting that neither the TCR constant domains nor potential N- or O-linked carbohydrate moieties are necessary for ligand recognition and for expression and proper folding of the D10 scTCR. Purified D10 scTCR remained soluble at concentrations up to 1 mM. Circular dichroism and NMR spectroscopy indicated that D10 scTCR is stabilized predominantly by beta-sheet secondary structure, consistent with its native-like conformation. Because of its limited size, high solubility, and structural integrity, purified D10 scTCR appears to be suitable for structural studies by multidimensional NMR spectroscopy.


Subject(s)
Histocompatibility Antigens Class II/metabolism , Receptors, Antigen, T-Cell/metabolism , Superantigens/metabolism , Amino Acid Sequence , Chromatography, Gel , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Kinetics , Ligands , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Peptides/metabolism , Protein Conformation , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
9.
Antimicrob Agents Chemother ; 40(1): 234-6, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8787913

ABSTRACT

PIC 024-4 and PRO 2000 are naphthalene sulfonate polymers that bind to CD4 with nanomolar affinity and block binding of gp120. Both have activity against human immunodeficiency virus type 1 in H9 cells, peripheral blood mononuclear cells, and primary monocyte/macrophages, are synergistic with zidovudine, and do not inhibit tetanus toxoid-stimulated T-cell proliferation at anti-human immunodeficiency virus type 1 concentrations.


Subject(s)
Antiviral Agents/pharmacology , CD4 Antigens/drug effects , HIV Envelope Protein gp120/drug effects , HIV-1/drug effects , Naphthalenesulfonates/pharmacology , Animals , Binding, Competitive , CD4-Positive T-Lymphocytes/drug effects , Cell Line, Transformed , Humans , Macaca fascicularis , Naphthalenesulfonates/toxicity , Polymers/pharmacology , Protein Binding/drug effects , Rats , Recombinant Proteins/drug effects
10.
Science ; 264(5155): 82-5, 1994 Apr 01.
Article in English | MEDLINE | ID: mdl-7511253

ABSTRACT

The crystal structure of a complex between a 24-amino acid peptide from the third variable (V3) loop of human immunodeficiency virus-type 1 (HIV-1) gp 120 and the Fab fragment of a broadly neutralizing antibody (59.1) was determined to 3 angstrom resolution. The tip of the V3 loop containing the Gly-Pro-Gly-Arg-Ala-Phe sequence adopts a double-turn conformation, which may be the basis of its conservation in many HIV-1 isolates. A complete map of the HIV-1 principal neutralizing determinant was constructed by stitching together structures of V3 loop peptides bound to 59.1 and to an isolate-specific (MN) neutralizing antibody (50.1). Structural conservation of the overlapping epitopes suggests that this biologically relevant conformation could be of use in the design of synthetic vaccines and drugs to inhibit HIV-1 entry and virus-related cellular fusion.


Subject(s)
Antigen-Antibody Complex/chemistry , HIV Antibodies/chemistry , HIV Envelope Protein gp120/chemistry , HIV-1/chemistry , Immunoglobulin Fab Fragments/chemistry , Peptide Fragments/chemistry , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antigen-Antibody Complex/immunology , Antigen-Antibody Reactions , Computer Graphics , Crystallography, X-Ray , Epitopes/chemistry , Epitopes/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Hydrogen Bonding , Immunoglobulin Fab Fragments/immunology , Models, Molecular , Molecular Sequence Data , Neutralization Tests , Peptide Fragments/immunology , Protein Conformation , Protein Structure, Secondary
11.
Structure ; 1(2): 83-93, 1993 Oct 15.
Article in English | MEDLINE | ID: mdl-8069628

ABSTRACT

BACKGROUND: Recent structural results have shown that antibodies use an induced fit mechanism to recognize and bind their antigens. Here we present the crystallographically determined structure of an Fab directed against an HIV-1 peptide (Fab 50.1) in the unliganded state and compare it with the peptide-bound structure. We perform a detailed analysis of the components that contribute to enhanced antigen binding and recognition. RESULTS: Induced fit of Fab 50.1 to its peptide antigen involves a substantial rearrangement of the third complementarity determining region loop of the heavy chain (H3), as well as a large rotation of the variable heavy (VH) chain relative to the variable light (VL) chain. Analysis of other Fab structures suggests that the extent of the surface area buried at the VL-VH interface correlates with the ability to alter antibody quaternary structure by reorientation of the VL-VH domains. CONCLUSION: Fab 50.1 exhibits the largest conformational changes yet observed in a single antibody. These can be attributed to the flexibility of the variable region. Comparisons of new data with previous examples lend to the general conclusion that a small VL-VH interface, due in part to a short H3 loop, permits substantial alterations to the antigen-binding pocket. This has major implications for the prediction, engineering and design of antibody-combining sites.


Subject(s)
HIV Antigens/immunology , Immunoglobulin Fab Fragments/chemistry , Protein Conformation , Protein Structure, Secondary , Viral Proteins/immunology , Amino Acid Sequence , Computer Graphics , Crystallography, X-Ray/methods , HIV-1 , Models, Molecular , Molecular Sequence Data
12.
Proc Natl Acad Sci U S A ; 90(13): 6325-9, 1993 Jul 01.
Article in English | MEDLINE | ID: mdl-8327513

ABSTRACT

The crystal structure of the Fab fragment of a human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibody Fab has been determined at 2.8 A resolution in complex with a linear 16-residue peptide from the third hypervariable region (V3) of gp120. The first 9 residues of the peptide are ordered in the electron density maps, and their conformation is in partial agreement with the beta-strand-type II beta-turn structure predicted for this portion of the V3 loop. Notably, several of the peptide residues that are well conserved among different HIV-1 isolates contact a nonpolar 25-A-long groove in the antibody-combining site. The largely extended structure of the peptide differs from the beta-turns seen as the primary determinants in other published anti-peptide Fab structures. Analysis of the specific Fab-peptide interactions only partially explains the MN isolate specificity shown by this antibody.


Subject(s)
Antibodies, Monoclonal/chemistry , HIV Antibodies/chemistry , HIV Envelope Protein gp120/chemistry , HIV-1/immunology , Immunoglobulin Fab Fragments/chemistry , Peptide Fragments/chemistry , Amino Acid Sequence , Animals , Computer Graphics , Crystallization , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Sequence Data , Protein Conformation , X-Ray Diffraction
13.
Proteins ; 14(4): 499-508, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1438187

ABSTRACT

X-ray quality crystals of an Fab fragment from an antipeptide monoclonal antibody (R/V3-50.1) that recognizes the principal neutralizing determinant (PND) of the gp120 glycoprotein of human immunodeficiency virus type 1 (HIV-1) (MN isolate) were grown as uncomplexed and peptide complexed forms. Crystals of the free Fab grew from high salt in orthorhombic space groups P2(1)2(1)2(1) and I222 and from polyethylene glycol in space groups P1 and P2(1). Seeds from either the P1 and P2(1) native (uncomplexed) Fab crystals induced nucleation of crystals of the Fab complexed to a 16-residue synthetic peptide corresponding to the PND when streak seeded into preequilibrated solutions of this complex. Data were collected from these complex crystals and from each of the four native Fab forms to at least 2.8 A resolution. The genes for the variable domain of the Fab were cloned and sequenced and the primary amino acid sequence was deduced from this information. Knowledge of the three-dimensional structure of this Fab-peptide complex will be important in the understanding of the PND of HIV-1 and its recognition by neutralizing monoclonal antibodies.


Subject(s)
Antibodies, Viral/chemistry , Antigen-Antibody Complex/chemistry , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Immunoglobulin Fab Fragments/chemistry , Peptide Fragments/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Viral/immunology , Antigen-Antibody Complex/immunology , Base Sequence , Crystallization , DNA, Single-Stranded , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Variable Region/chemistry , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Neutralization Tests , Solubility
14.
J Immunol ; 148(3): 914-20, 1992 Feb 01.
Article in English | MEDLINE | ID: mdl-1370524

ABSTRACT

Multiple Ag peptide (MAP) system without the use of a protein carrier was used as a vaccine model in three species of animals. Synthetic peptides from the V3 region of the gp120 of IIIB, RF and MN HIV-1 isolates were used as the Ag. MAP consisting of various chain lengths, from 11 to 24 residues, were prepared in a monoepitope configuration containing four repeats of each individual peptide. In parallel, they were synthesized in a diepitope configuration adding at the carboxyl-terminus of the V3 peptides a conserved sequence, known to be a Th cell epitope of gp120. The antibody response elicited by the monoepitope constructs was species-dependent. Rabbits produced immunity against all nine peptides, whereas mice were strongly reactive mainly to the longest sequence of the IIIB isolate. The immune response of guinea pigs was intermediate to those of rabbits and mice. Diepitope MAPs were immunogenic in all three species and elicited significantly higher titers than those raised by the immunization with the monoepitope MAPs. The response was type specific; the high-titered antibodies were reactive mostly against the isolate from which the peptides were derived, with a small cross-reactivity in ELISA between IIIB and RF strains. The dominant antigenic site of the B cell epitope, IIIB sequence, was located at the amino and central part of the MAP and a sequence overlapping the putative V3 reverse-turn was particularly reactive with the raised antibodies. Moreover, sera from the immunized animals inhibited virus-dependent cell fusion. These results show that MAP, with a chemically defined structure and without the use of a protein carrier, can be potentially useful for the design of synthetic HIV-1 vaccine candidates.


Subject(s)
B-Lymphocytes/immunology , HIV Antibodies/biosynthesis , HIV Antigens/chemistry , HIV-1/immunology , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes/immunology , Vaccines, Synthetic/chemistry , Amino Acid Sequence , Animals , Epitopes , Guinea Pigs , Lymphocyte Activation , Mice , Molecular Sequence Data , Rabbits , Vaccines, Synthetic/immunology
15.
Biochemistry ; 30(38): 9187-94, 1991 Sep 24.
Article in English | MEDLINE | ID: mdl-1892828

ABSTRACT

With standard one- and two-dimensional proton NMR techniques, a common structural motif has been identified in water solutions of short peptide sequences derived from the envelope glycoprotein gp120 of HIV-1. Three peptides of lengths 12, 24, and 40 residues (termed RP342, RP142, and RP70, respectively) were synthesized, each containing a central amino acid sequence common to many HIV-1 isolates. In addition, RP70 contained a disulfide bond between cysteine residues close to the ends of the molecule, forming a loop that is thought to constitute an important structural and immunological component of the intact glycoprotein. Peptides RP70 and RP142 showed evidence for the presence of a significant population of conformations containing a beta-turn in the conserved sequence Gly-Pro-Gly-Arg. Strong nuclear Overhauser effect (NOE) connectivities were observed between the amide protons of the arginine and the adjacent glycine. A weak NOE connectivity was observed between the C alpha H of the proline residue and the NH of the Arg [a d alpha N(i,i + 2) NOE connectivity], confirming the presence of a conformational preference for a turn conformation in this sequence. The remainder of the peptide showed evidence of conformational averaging: no NMR evidence for a uniquely folded structure was obtained for any of the peptides in water solution. Circular dichroism (CD) spectra indicated that no ordered helix was present in water solutions of RP70, although a CD spectrum that indicated the presence of approximately 30% helix could be induced by the addition of trifluoroethanol.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
HIV Antigens/chemistry , HIV Envelope Protein gp120/chemistry , HIV-1/immunology , Amino Acid Sequence , Circular Dichroism , Disulfides/chemistry , HIV Envelope Protein gp120/immunology , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Peptides/chemistry , Protein Conformation , Solutions , Solvents , Temperature
17.
J Immunol Methods ; 142(1): 53-9, 1991 Aug 28.
Article in English | MEDLINE | ID: mdl-1919021

ABSTRACT

Immunoassays designed to measure low concentrations of staphylococcal protein A are subject to varying degrees of interference by excess IgG. We have developed an enzyme-linked immunosorbent assay (ELISA) that overcomes this problem by analyzing IgG-containing protein A samples in solutions buffered at pH 3.5. Under these carefully selected conditions, protein A is absorbed efficiently by solid-phase chicken anti-protein A antibodies, but protein A-IgG complexes are largely dissociated. The assay has a protein A detection limit of 0.1 ng/ml, and the response is unaffected by 0.25 mg/ml murine IgG. The method should be useful for determining protein A contamination levels in antibodies purified by affinity chromatography on immobilized protein A resins.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin G/chemistry , Staphylococcal Protein A/analysis , Animals , Chickens , Chromatography, Ion Exchange , Hydrogen-Ion Concentration , Protein Binding , Recombinant Proteins , Reproducibility of Results , Staphylococcal Protein A/isolation & purification
19.
Science ; 250(4987): 1590-3, 1990 Dec 14.
Article in English | MEDLINE | ID: mdl-1703322

ABSTRACT

The principal neutralizing determinant (PND) of human immunodeficiency virus (HIV)-1 resides within the V3 loop of the envelope protein. Antibodies elicited by peptides of this region were able to neutralize diverse isolates. Serum from one of three animals immunized with the human T cell lymphoma virus (HTLV)-IIIMN PND peptide, RP142, neutralized MN and the sequence-divergent HTLV-IIIB isolate. Serum from one of three animals immunized with a 13-amino acid IIIB PND peptide (RP337) also neutralized both of these isolates. Characterization of these sera revealed that the cross-neutralizing antibodies bound the amino acid sequence GlyProGlyArgAlaPhe (GPGRAF) that is present in both isolates. This sequence is frequently found in the PNDs analyzed in randomly selected HIV-1 isolates. Sera from two rabbits immunized with a peptide containing only the GPGRAF residues neutralized divergent isolates, including IIIB and MN.


Subject(s)
Epitopes/immunology , HIV Antibodies/immunology , HIV Antigens/immunology , HIV-1/immunology , Acquired Immunodeficiency Syndrome/microbiology , Amino Acid Sequence , Animals , Enzyme-Linked Immunosorbent Assay , Guinea Pigs , Humans , Immune Sera/immunology , Immunization , Molecular Sequence Data , Neutralization Tests , Rabbits , Viral Envelope Proteins/immunology
20.
Proc Natl Acad Sci U S A ; 87(21): 8597-601, 1990 Nov.
Article in English | MEDLINE | ID: mdl-1700435

ABSTRACT

We describe a human IgG1 monoclonal antibody (N701.9b) derived by Epstein-Barr virus transformation of B cells from a human immunodeficiency virus-seropositive asymptomatic donor. This antibody was shown to recognize the principal neutralizing domain contained within the V3 region of gp120 of the MN strain of human immunodeficiency virus and MN-like strains, as determined by binding to the PB-1 fragment of MN gp120 and to synthetic peptides corresponding to the V3 region of MN and related virus strains. The epitope identified by monoclonal antibody N701.9b was mapped to a segment of V3 containing at least 7 amino acids (amino acids 316-322), which is located in the "tip" and "right" side of the V3 loop of the MN strain. Furthermore, this antibody manifested potent type-specific fusion-inhibitory activity against the MN strain but not against the IIIB or RF virus strains. This antibody also neutralized four virus isolates that had MN-like V3 region sequences and failed to neutralize three other strains containing unrelated V3 region sequences. Our findings confirm that the V3 region stimulates type-specific neutralizing antibody during natural human immunodeficiency virus infection in humans. The potential clinical use of this antibody is discussed.


Subject(s)
Antibodies, Monoclonal/immunology , Antibody-Dependent Cell Cytotoxicity , Epitopes/analysis , HIV Envelope Protein gp120/immunology , HIV-1/immunology , HIV/immunology , Amino Acid Sequence , B-Lymphocytes/immunology , Epitopes/immunology , HIV/genetics , HIV Envelope Protein gp120/genetics , HIV-1/isolation & purification , Humans , Molecular Sequence Data , Neutralization Tests , Species Specificity , Vaccinia virus/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...