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1.
Diabetes Obes Metab ; 12(11): 983-93, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20880345

ABSTRACT

AIMS: To assess the efficacy and safety of ezetimibe/simvastatin (E/S) plus extended-release niacin (N) in hyperlipidaemic patients with diabetes mellitus (DM), metabolic syndrome (MetS) without DM (MetS/non-DM) or neither (non-DM/non-MetS). METHODS: A subgroup analysis of a double-blind, 64-week trial of 1220 randomized patients who received E/S (10/20 mg) + N (to 2 g) or E/S (10/20 mg) for 64 weeks, or N (to 2 g) for 24 weeks then E/S (10/20 mg) + N (2 g) or E/S (10/20 mg) for 40 additional weeks. The evaluable populations of this analysis included n = 765 patients at 24 weeks and n = 574 at 64 weeks. Among those receiving N, only those who attained the 2-g dose were included in the analysis. RESULTS: E/S+N improved levels of low-density lipoprotein cholesterol, other lipids and lipoprotein ratios compared with N and E/S at 24 weeks and E/S at 64 weeks. The combination increased high-density lipoprotein cholesterol and apolipoprotein AI comparably to N and more than E/S. E/S+N reduced high-sensitivity C-reactive protein (hsCRP) levels more effectively than N and similarly to E/S. E/S+N was generally well tolerated. Discontinuations due to flushing with N and E/S+N were comparable and greater than E/S in all subgroups. Fasting glucose trended higher for N vs. E/S. Glucose elevations from baseline to 12 weeks were highest for patients with DM (24.9 mg/dl for N, 21.2 mg/dl for E/S+N, 17.5 mg/dl for E/S); fasting glucose then declined to pretreatment levels at 64 weeks in all subgroups. New-onset DM was more frequent among MetS patients than those without MetS during the first 24 weeks and trended higher among those assigned to N-containing regimens [n = 5(5.1%) for N, n = 2(1.7%) for E/S, n = 21(8.8%) for E/S+N]; during the 24-64 week extension study, diabetes was diagnosed in five additional patients in the E/S(cumulative incidence of 5.9%) and one in the E/S+N (cumulative incidence of 9.2%). Treatment-incident elevations in uric acid levels were increased among subjects assigned to N-containing regimens, but there were no effects on symptomatic gout. CONCLUSION: Combination E/S+N is a safe treatment option for hyperlipidaemic patients including those with DM and MetS, but requires monitoring of glucose and potentially uric acid levels.


Subject(s)
Anticholesteremic Agents/administration & dosage , Azetidines/administration & dosage , Delayed-Action Preparations , Diabetes Mellitus, Type 2/drug therapy , Hyperlipidemias/drug therapy , Metabolic Syndrome/drug therapy , Niacin/administration & dosage , Simvastatin/administration & dosage , Cholesterol, LDL/drug effects , Drug Combinations , Ezetimibe, Simvastatin Drug Combination , Female , Humans , Male , Middle Aged , Treatment Outcome
2.
Diabetes Obes Metab ; 11(9): 855-64, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19508464

ABSTRACT

AIM: To evaluate the effects of the usual starting and next higher doses of ezetimibe/simvastatin and atorvastatin on the cholesterol content of lipoprotein subclasses in patients with type 2 diabetes and hypercholesterolaemia. METHODS: This post hoc analysis compared the effects of treatment with ezetimibe/simvastatin 10/20 mg vs. atorvastatin 10 and 20 mg/day and ezetimibe/simvastatin 10/40 mg/day vs. atorvastatin 40 mg/day on the cholesterol content of lipoprotein subclasses in the modified intent-to-treat (mITT) population (n = 1013) and in subgroups of patients with triglyceride (TG) levels <200 mg/dl (n = 600) and >or=200 mg/dl (2.6 mmol/l) (n = 413). RESULTS: Ezetimibe/simvastatin significantly reduced low-density lipoprotein cholesterol (LDL-C) subclasses LDL(1)-C, LDL(2)-C and LDL(3)-C; real LDL-C (LDL-C(r)); intermediate-density lipoprotein cholesterol (IDL-C), IDL(1)-C, IDL(2)-C; very low-density lipoprotein cholesterol (VLDL-C), VLDL(3)-C; and remnant-like lipoprotein cholesterol (RLP-C) from baseline more than atorvastatin at all dose comparisons (p < 0.01) in the mITT population. Significant improvements were also observed in high-density lipoprotein cholesterol (HDL-C) subclass HDL(3)-C at the ezetimibe/simvastatin 10/20 mg vs. atorvastatin 20 mg and highest dose comparisons (p < 0.001) and in VLDL(1 + 2)-C at the lowest and highest dose comparisons (p < 0.001). Changes in LDL(4)-C and LDL-C subclass patterns (A, B and I) were comparable for both treatments. Generally, similar results were observed for patients with TG levels <200 and >or=200 mg/dl (2.3 mmol). For both treatments, notable differences between TG subgroups were that patients with elevated TGs had smaller reductions in LDL(2)-C, slightly smaller decreases in all IDL subclasses and greater decreases in all VLDL-C subclasses than those with lower TG levels. Frequency of pattern B was also reduced more in patients with higher TGs for both treatments. CONCLUSIONS: Ezetimibe/simvastatin reduced the cholesterol content of most lipoprotein subclasses from baseline with generally similar efficacy in patients with low and high TGs. Despite the different mechanism of action of ezetimibe, the response to ezetimibe/simvastatin and atorvastatin treatment related to these lipoprotein subclasses was generally consistent with the overall effects of these therapies on the major lipid/lipoprotein classes. The clinical significance of these results awaits further study.


Subject(s)
Anticholesteremic Agents/administration & dosage , Azetidines/administration & dosage , Diabetes Mellitus, Type 2/drug therapy , Heptanoic Acids/administration & dosage , Hypercholesterolemia/drug therapy , Pyrroles/administration & dosage , Simvastatin/administration & dosage , Adult , Aged , Atorvastatin , Cholesterol/blood , Cholesterol/classification , Cholesterol, HDL/blood , Cholesterol, LDL/blood , Cholesterol, VLDL/blood , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/complications , Drug Therapy, Combination , Ezetimibe , Female , Humans , Hypercholesterolemia/blood , Hypercholesterolemia/complications , Male , Middle Aged , Young Adult
3.
Virology ; 313(1): 274-85, 2003 Aug 15.
Article in English | MEDLINE | ID: mdl-12951039

ABSTRACT

We have established an in vitro replication system for bovine viral diarrhea virus (BVDV), a surrogate for the closely-related hepatitis C virus. In an in vitro reaction, BVDV replication complexes synthesize vRNA and replicative form (RF) and replicative intermediate (RI) RNAs. Kinetic and heparin trapping experiments demonstrate the recycling of RF and RI products and the initiation of vRNA synthesis in this system. Consistent with this, quantitative hybridization reveals the asymmetric synthesis of positive and negative strand RNA products. These findings support the notion that RF serves as a template and RI as a precursor in the synthesis of vRNA. Furthermore, the antiviral activity of an NS5B inhibitor was similar in BVDV replicase and infectivity assays. Together, these results indicate that the in vitro activity of BVDV replicase complexes recapitulates RNA replication that occurs in infected cells, providing a system in which to study both mechanisms and inhibitors of Flaviviridae replication.


Subject(s)
Diarrhea Viruses, Bovine Viral/physiology , RNA, Viral/biosynthesis , RNA-Dependent RNA Polymerase/metabolism , Animals , Cattle , Cell Line , Diarrhea Viruses, Bovine Viral/enzymology , Diarrhea Viruses, Bovine Viral/genetics , Enzyme Inhibitors/pharmacology , Heparin/pharmacology , RNA, Viral/chemistry , RNA, Viral/genetics , Subcellular Fractions/metabolism , Time Factors , Viral Nonstructural Proteins/antagonists & inhibitors , Virus Replication/drug effects
4.
J Org Chem ; 66(16): 5504-16, 2001 Aug 10.
Article in English | MEDLINE | ID: mdl-11485475

ABSTRACT

Flutimide, a fully substituted 1-hydroxy-3H-pyrazine-2,6-dione, is a fungal metabolite isolated from a new species of Delitschia cofertaspora. It has been shown to selectively inhibit cap-dependent endonuclease activity of influenza virus A. The inhibition of this activity is a target for the potential development of a therapeutic agent to treat influenza infections. A convergent total synthesis of flutimide starting from L-leucine has been described. The synthetic methodology has been extended to include the synthesis of specifically designed aromatic analogues of flutimide, some of which exhibited greater than 7-fold improvement in activity. The most potent compounds were those with p-fluorobenzylidene or p-methoxybenzylidene substitutions at C-5 of 3H-pyrazine-2,6-dione and showed IC(50) values of 0.9 and 0.8 microM, respectively. The details of the rationale for the synthetic design, syntheses, and biological activities of these analogues are described.


Subject(s)
Antiviral Agents/chemical synthesis , Endonucleases/antagonists & inhibitors , Orthomyxoviridae/enzymology , Piperazines/chemical synthesis , Pyrazines/chemical synthesis , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Inhibitory Concentration 50 , Orthomyxoviridae/drug effects , Oxidation-Reduction , Piperazines/chemistry , Piperazines/pharmacology , Pyrazines/chemistry , Pyrazines/pharmacology
5.
Antimicrob Agents Chemother ; 40(5): 1189-93, 1996 May.
Article in English | MEDLINE | ID: mdl-8723464

ABSTRACT

A novel anti-influenza virus compound, flutimide, was identified in extracts of a recently identified fungal species, Delitschia confertaspora (F. Pelaez, J.D. Polishook, M. Valldosera, and J.Guarro, Mycotaxon 50:115-122, 1994). The compound, a substituted 2,6-diketopiperazine, selectively inhibited the cap-dependent transcriptase of influenza A and B viruses and had no effect on the activities of other polymerases. Similar to the 4-substituted 2,4-dioxobutanoic acids, a series of transcriptase inhibitors which we described previously (J. Tomassini, H. Selnick, M.E. Davies, M.E. Armstrong, J. Baldwin, M. Bourgeois, J.Hastings, D. Hazuda, J. Lewis, W. McClements, G. Ponticello, E. Radzilowski, G. Smith, A. Tebben, and A. Wolfe, Antimicrob. Agents Chemother. 38:2827-2837, 1994), this inhibitor, which is a natural product, affected neither the initiation nor the elongation of influenza virus mRNA synthesis, but it specifically targeted the cap-dependent endonuclease of the transcriptase. Additionally, the compound was inhibitory to the replication of influenza A and B viruses in cell culture. The selective antiviral properties of this compound further demonstrate the utility of influenza virus endonuclease as a target of antiviral agents.


Subject(s)
Antiviral Agents/pharmacology , DNA-Directed RNA Polymerases/antagonists & inhibitors , Influenza A virus/drug effects , Influenza B virus/drug effects , Piperazines/pharmacology , Ascomycota/chemistry , Cells, Cultured , DNA-Directed RNA Polymerases/metabolism , Influenza A virus/enzymology , Influenza B virus/enzymology , Piperazines/chemistry , Piperazines/isolation & purification , RNA Caps/metabolism , RNA, Viral/biosynthesis , Structure-Activity Relationship , Transcription, Genetic/drug effects , Virus Replication/drug effects
6.
Antimicrob Agents Chemother ; 40(5): 1304-7, 1996 May.
Article in English | MEDLINE | ID: mdl-8723491

ABSTRACT

We previously identified a series of compounds which specifically inhibited the transcription of influenza A and B viruses (J. Tomassini, H. Selnick, M.E. Davies, M.E. Armstrong, J. Baldwin, M. Bourgeois, J. Hastings, D. Hazuda, J. Lewis, W. McClements, G. Ponticello, E. Radzilowski, G. Smith, A. Tebben, and A. Wolfe, Antimicrob. Agents Chemother. 38:2827-2837, 1994). The compounds, 4-substituted 2,4-dioxobutanoic acids, selectively targeted the cap-dependent endonuclease activity of the transcriptase complex. Additionally, several of these compounds effectively inhibited the replication of influenza virus but not other viruses in cell culture assays. Here, we report on the anti-influenza virus activities of other potent derivatives of the series evaluated in both in vitro and in vivo infectivity assays. These compounds inhibited the replication of influenza virus in yield reduction assays, with 50% inhibitory concentrations ranging from 0.18 to 0.71 microM. These 50% inhibitory concentrations were similar to those observed for inhibition of in vitro transcription (0.32 to 0.54 microM). One selected compound also elicited a dose-dependent inhibition of influenza virus replication in mice following an upper respiratory tract challenge. These studies demonstrate the antiviral efficacy of this inhibitor class and thereby establish the utility of influenza virus endonuclease as a chemotherapeutic target.


Subject(s)
Antiviral Agents/pharmacology , Butyrates/pharmacology , Orthomyxoviridae/drug effects , Animals , Cells, Cultured , Endoribonucleases/antagonists & inhibitors , Mice , Orthomyxoviridae/enzymology , Transcription, Genetic/drug effects , Virus Replication/drug effects
8.
J Virol ; 64(6): 2582-7, 1990 Jun.
Article in English | MEDLINE | ID: mdl-1970837

ABSTRACT

The vast majority of human rhinovirus serotypes utilize the intercellular adhesion molecule 1 (ICAM-1) as the attachment site on susceptible cells. Twelve murine monoclonal antibodies were isolated and shown by competition binding studies to recognize three distinct, nonoverlapping epitopes on the ICAM-1 receptor. Titration of three antibodies representing each of the binding sites demonstrated that they were equally effective at blocking viral attachment. By using in vitro transcription and translation systems, a series of progressive C-terminal truncations of ICAM-1 molecules was generated. Immunoprecipitation of these fragments with each of the three antibodies indicated that all three epitopes reside within the first 82 amino acids of the receptor. Attempts to demonstrate specific binding of these in vitro-synthesized receptor fragments to virions were unsuccessful. The inability to show virion binding was most likely due to a failure of the lysates to properly glycosylate the receptor molecule, since native, unglycosylated receptor molecules isolated from cell membranes were also inactive in virus binding assays.


Subject(s)
Antibodies, Monoclonal/immunology , Cell Adhesion Molecules/physiology , Receptors, Virus/physiology , Rhinovirus/physiology , Animals , Antigen-Antibody Complex , Base Sequence , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/immunology , Humans , Intercellular Adhesion Molecule-1 , Kinetics , Mice , Mice, Inbred BALB C/immunology , Molecular Sequence Data , Oligonucleotide Probes , Polymerase Chain Reaction , Protein Biosynthesis , Receptors, Virus/immunology , Restriction Mapping , Rhinovirus/immunology , Transcription, Genetic
9.
J Biol Chem ; 265(4): 2292-5, 1990 Feb 05.
Article in English | MEDLINE | ID: mdl-2153680

ABSTRACT

Human rhinoviruses are the major causative agents of the common cold in humans and have been divided into major and minor groups based on receptor specificity. cDNAs encoding the light and heavy chains of a murine monoclonal antibody that recognizes the major group receptor were cloned, abundantly expressed in Escherichia coli, and renatured into Fab fragments that blocked virus binding and protected HeLa cell monolayers from rhinovirus infection. Elimination of the cysteines normally bridging the heavy and light chains yielded molecules indistinguishable from wild-type Fab fragments in virus binding assays. Single-chain antibodies with covalently linked light and heavy variable domains were also expressed and showed receptor binding and cell protection activities. These recombinant antibody fragments are potentially useful in preventing or treating common colds in humans.


Subject(s)
Genes, Immunoglobulin , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/immunology , Receptors, Virus/physiology , Rhinovirus/immunology , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Cell Transformation, Viral , Cloning, Molecular , Escherichia coli/genetics , Gene Expression , HeLa Cells/physiology , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/isolation & purification , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/isolation & purification , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/isolation & purification , Molecular Sequence Data , Protein Conformation , Receptors, Virus/immunology , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Rhinovirus/genetics , Rhinovirus/physiology
10.
Proc Natl Acad Sci U S A ; 86(13): 4907-11, 1989 Jul.
Article in English | MEDLINE | ID: mdl-2544880

ABSTRACT

A 90-kDa surface glycoprotein was previously isolated and shown to be required for infection by the "major" group of human rhinovirus (HRV) serotypes. In the present work, the amino acid sequence of the receptor protein was obtained from CNBr and tryptic peptides. Using degenerate oligonucleotides predicted from the peptide sequences, we identified four cDNA clones that encode a 3-kilobase mRNA. The clones were ligated, subcloned in a simian virus 40 expression vector, and used to transfect receptor-negative Vero (monkey) cells. Results showed that transfected cells expressed receptor molecules capable of binding HRV and a monoclonal antibody which recognizes the major group HRV receptor. The cloned receptor cDNA encoded a protein with a sequence nearly identical to that of the intercellular adhesion molecule 1 (ICAM-1), indicating that the two surface proteins are one and the same. Both proteins have identical mass, carbohydrate composition, and tissue distribution. In addition, major group receptors on HeLa cells could be induced with various cytokines in a manner similar to the ICAM-1 ligand. A similar induction of the HRV "minor" group receptor was not observed.


Subject(s)
Antigens, Surface/genetics , Cloning, Molecular , Genes , Receptors, Virus/genetics , Rhinovirus/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Adhesion , Cell Adhesion Molecules , DNA, Neoplasm/genetics , HeLa Cells/immunology , HeLa Cells/metabolism , Humans , Molecular Sequence Data , Peptide Fragments/isolation & purification , Sequence Homology, Nucleic Acid , Transfection , Vero Cells
11.
J Virol ; 63(1): 36-42, 1989 Jan.
Article in English | MEDLINE | ID: mdl-2535737

ABSTRACT

Previous molecular and immunological studies have mapped four neutralization sites on human rhinovirus type 14 (B. Sherry, A. G. Mosser, R. J. Colonno, and R. R. Rueckert, J. Virol. 57:246-257, 1986). Eight monoclonal antibodies, one pair for each of the four target sites and all belonging to a single isotype, immunoglobulin G2a, were studied under conditions which resulted in 95% neutralization of infectious viral particles. All eight antibodies shifted the isoelectric point of virions from 6.7 to much more acidic forms, ranging from pI 1.8 to 3.2. In addition, antibodies targeted against three of the four neutralization sites caused significant aggregation of virions under the neutralization conditions employed. Aggregation could be reversed by digesting virus-antibody complexes with papain. Following papain digestion, the acidic pIs of three of the neutralized virus preparations returned to neutral and infectivity was restored. Membrane-binding assays with virus neutralized with a nonaggregating antibody showed a dose-related inhibition of virus attachment to cellular receptors. Purified Fab fragments at a 13- to 61-fold-higher concentration than intact antibodies caused a comparable isoelectric shift, neutralized virions in the absence of aggregation, and interfered with attachment of virions to host cell receptors in a membrane-binding assay. These findings suggest that neutralizing antibodies interfere with the attachment of rhinoviruses to cellular receptors and that bivalent attachment of antibody is not a prerequisite for neutralization.


Subject(s)
Antibodies, Monoclonal/immunology , Immunoglobulin Fab Fragments/immunology , Rhinovirus/immunology , Binding Sites, Antibody , Cell Membrane/microbiology , Electrophoresis, Polyacrylamide Gel , HeLa Cells , Humans , Isoelectric Focusing , Neutralization Tests , Papain/pharmacology , Rhinovirus/drug effects , Rhinovirus/metabolism , Virion/drug effects , Virion/metabolism
12.
J Biol Chem ; 264(3): 1656-62, 1989 Jan 25.
Article in English | MEDLINE | ID: mdl-2536369

ABSTRACT

Human rhinoviruses attach to specific receptors located on the surfaces of host cells as a first step in viral infection. A 90-kDa cell surface protein was previously shown to be involved in the attachment of human rhinoviruses to susceptible cells (Tomassini, J. E., and Colonno, R.J. (1986) J. Virol. 58, 290-295). Digestion of purified receptor protein with various glycosidases revealed that 30% of its molecular mass was comprised of complex-type oligosaccharides, one-third being contributed by sialic acid. The presence of sialic acid was confirmed by demonstrating that wheat germ lectin can inhibit the attachment of rhinoviruses to host cell membranes, while lectins of other sugar specificities had no effect. The oligosaccharides were shown to be N-linked by tunicamycin treatment of host cells and by N-glycanase digestion. Seven N-linked glycosylation sites were detected by partial digestion of the receptor oligosaccharides with N-glycanase. Native receptor protein had an isoelectric focusing point of 4.2, compared to 5.3 for the deglycosylated protein. Studies of virus and antibody binding to neuraminidase-treated host cell membranes suggested that although carbohydrates may be involved in host-virus interaction, the receptor carbohydrate is not the predominant component of the cellular receptor site.


Subject(s)
Attachment Sites, Microbiological , Lysogeny , Membrane Glycoproteins/analysis , Rhinovirus/metabolism , Carbohydrate Conformation , Glycoside Hydrolases/metabolism , Glycosylation , HeLa Cells/analysis , HeLa Cells/drug effects , Humans , Isoelectric Point , Lectins/pharmacology , Receptors, Virus/analysis , Sialic Acids/analysis , Tunicamycin/pharmacology
13.
J Virol ; 58(2): 290-5, 1986 May.
Article in English | MEDLINE | ID: mdl-3009846

ABSTRACT

Human rhinoviruses can be classified into major and minor groups on the basis of receptor specificity. Recently, a mouse monoclonal antibody was isolated which selectively blocked the attachment of the major group of human rhinoviruses to cells. Using this monoclonal antibody, the cellular receptor for the major group of human rhinoviruses was isolated. A radioimmunoassay was developed by using the receptor antibody to specifically detect rhinovirus receptor during isolation. Solubilized receptor from detergent-treated HeLa cell membrane extracts eluted from gel filtration columns with an apparent molecular weight of 440,000. A cellular receptor protein, which had a molecular weight of 90,000 when analyzed on sodium dodecyl sulfate-polyacrylamide gels, was purified from solubilized extracts on an immunoaffinity column containing receptor antibody. Polyclonal rabbit antiserum, resulting from immunization with the isolated receptor protein, specifically blocked the attachment of the major group of human rhinoviruses and indicated that the 90-kilodalton protein plays a functional role in attachment. Prolonged exposure of HeLa cell monolayers with the receptor antibody showed no inhibition of cell growth and division.


Subject(s)
Receptors, Virus/isolation & purification , Rhinovirus/metabolism , Cell Division , Cell Membrane/microbiology , Chromatography, Affinity , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , HeLa Cells , Humans , Immune Sera , Molecular Weight , Receptors, Virus/immunology , Receptors, Virus/physiology , Solubility
14.
J Virol ; 52(2): 465-73, 1984 Nov.
Article in English | MEDLINE | ID: mdl-6208377

ABSTRACT

BALB/c mice were immunized with purified preparations of hepatitis A virus (HAV) isolated after 21 days of growth in LLC-MK2 cells. The HAV antigen was isolated from CsCl gradients and consisted primarily of the following three proteins as analyzed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining: VP-1 at 33,000 daltons, VP-2 at 29,000 to 30,000 daltons, and VP-3 at 27,000 daltons. The spleen cells isolated from two BALB/c mice, immunized with two inoculations of HAV, were fused with SP 2/0 myeloma cells and grown in hypoxanthine-aminopterin-thymidine medium. Of 270 hybridomas initially screened, 72 were positive for binding HAV by a noncompetitive radioimmunoassay. All 72 were tested for the ability to neutralize the infectivity of HAV in an in vitro cell culture assay that was adapted for microtiter plates and that used detergent-treated virus for improved neutralization sensitivity and newborn cynomolgus monkey kidney cells for rapid growth. Eighteen hybridomas were positive for neutralization; 16 remained stable. Of the 16, 9 were able to compete with labeled polyclonal serum for binding to HAV. The nine competing hybridomas could be separated into two groups which appear to be directed towards two different sites on HAV and could complement each other in the competitive radioimmunoassay against polyclonal sera. Of the original 16 neutralizing hybridomas, 4 were subcloned through two cycles of limit dilutions. All four monoclonal antibodies retained their original neutralizing and competitive properties; three were immunoglobulin G2a, and one was immunoglobulin G1. All four monoclonal antibodies readily precipitate whole 125I-labeled HAV but are not able to recognize the disrupted proteins of the virus (as tested by immune precipitations of heat- and detergent-disrupted virions or Western blot analyses). However, the heterobifunctional cross-linking reagent toluene-2,4-diisocyanate was used to cross-link purified Fab fragments of two different monoclonal antibodies (2D2 and 6A5) to HAV before disruption. This reagent demonstrated a specific reaction of the monoclonal antibodies to the VP-1 of HAV, suggesting this major surface protein contains at least one of the major neutralization sites for HAV.


Subject(s)
Epitopes/analysis , Hepatovirus/immunology , Viral Proteins/analysis , Animals , Antibodies, Monoclonal , Antigen-Antibody Complex , Cell Line , Hybridomas/immunology , Molecular Weight , Radioimmunoassay , Viral Proteins/immunology
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