34 results on '"Randal A. Byrn"'
Search Results
2. Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro
- Author
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Sudhir Agrawal, Randal A. Byrn, and Gareth J. Veal
- Subjects
Messenger RNA ,biology ,RNase P ,Oligonucleotide ,Ribonuclease H ,Gene Products, gag ,HIV Infections ,Nuclease protection assay ,U937 Cells ,Oligonucleotides, Antisense ,Virus Replication ,Cleavage (embryo) ,Molecular biology ,RNase MRP ,HIV-1 ,Genetics ,biology.protein ,Humans ,RNA, Viral ,RNA, Messenger ,Ribonuclease ,RNase H ,Sequence Analysis ,Research Article - Abstract
We have used a ribonuclease protection assay to investigate RNase H cleavage of HIV-1 mRNA mediated by phosphorothioate antisense oligonucleotides complementary to the gag region of the HIV-1 genome in vitro. Cell lysate experiments in H9 and U937 cells chronically infected with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no cleavage of spliced message which did not contain the target gag region. RNase H cleavage products were detected at oligonucleotide concentrations as low as 0.01 microM and the RNase H activity was seen to be concentration dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides demonstrated sequence specificity at low concentrations, with cleavage of gag mRNA correlating with the predicted activities of the parent and mismatch oligonucleotides based on their hybridization melting temperatures. Experiments in living cells suggested that RNase H-specific antisense activity was largely determined by the amount of oligonucleotide taken up by the different cell lines studied. RNase H cleavage products were detected in antisense oligonucleotide treated MT-4 cells acutely infected with HIV-1 IIIB, but not in infected H9 cells treated with oligonucleotide under the same conditions. The data presented demonstrate potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with previous reports that the major obstacle to demonstrating antisense activity in living cells remains the lack of penetration of these agents into the desired cellular compartment.
- Published
- 1998
3. The Multiple Inhibitory Mechanisms of GEM 91®, agagAntisense Phosphorothioate Oligonucleotide, for Human Immunodeficiency Virus Type 1
- Author
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Sudhir Agrawal, Ahmad N. Ali, Randal A. Byrn, Béla Papp, Koushi Yamaguchi, and Dezhen Zhang
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DNA Replication ,Transcription, Genetic ,Anti-HIV Agents ,viruses ,Immunology ,HIV Infections ,Biology ,Virus Replication ,Virus ,Oligodeoxyribonucleotides, Antisense ,Zidovudine ,Virion binding ,Viral entry ,Virology ,Gene expression ,medicine ,Humans ,RNA, Messenger ,Cells, Cultured ,Oligonucleotide ,Virus Assembly ,Drug Resistance, Microbial ,Oligonucleotides, Antisense ,Thionucleotides ,Blotting, Northern ,Molecular biology ,Reverse transcriptase ,Infectious Diseases ,Viral replication ,HIV-1 ,RNA, Viral ,medicine.drug - Abstract
GEM 91 (gene expression modulator) is a 25-mer oligonucleotide phosphorothioate complementary to the gag initiation site of HIV-1. GEM 91 has been studied in various in vitro cell culture models to examine inhibitory effects on different stages of HIV-1 replication. Experiments were focused on the binding of virions to the cell surface, inhibition of virus entry, reverse transcription (HIV DNA production), inhibition of steady state viral mRNA levels, inhibition of virus production from chronically infected cells, and inhibition of HIV genome packaging within virions. Experiments were also performed in vitro in an attempt to generate strains of HIV with reduced sensitivity to GEM 91. We observed sequence-dependent inhibition of virus entry/reverse transcription and a reduction in steady state viral RNA levels. We also observed sequence-independent inhibition of virion binding to cells and inhibition of virus production by chronically infected cells. Using in vitro methods that were successful in generating HIV strains with reduced sensitivity to AZT, we were unable to generate strains with reduced sensitivity to GEM 91.
- Published
- 1997
4. Mixed backbone antisense oligonucleotides: design, biochemical and biological properties of oligonucleotides containing 2'-5'-ribo- and 3'- 5'-deoxyribonucleotide segments
- Author
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Qiuyan Zhao, Sudhir Agrawal, Adrienne Manning, Viswanathan Sasisekharan, Denise R. Shaw, Ekambar R. Kandimalla, and Randal A. Byrn
- Subjects
Ribonuclease H ,Biology ,Nucleic Acid Denaturation ,DNA, Antisense ,Mice ,chemistry.chemical_compound ,Deoxyribonucleotide ,Genetics ,Animals ,RNA, Antisense ,RNase H ,Cells, Cultured ,Oligoribonucleotides ,Phosphoric Diester Hydrolases ,Oligonucleotide ,Circular Dichroism ,Single-Strand Specific DNA and RNA Endonucleases ,RNA ,Complement System Proteins ,Genes, gag ,Oligodeoxyribonucleotides ,Biochemistry ,chemistry ,Phosphodiesterase I ,Spectrophotometry ,Phosphodiester bond ,HIV-1 ,biology.protein ,Biophysics ,Nucleic acid ,Nucleic Acid Conformation ,Electrophoresis, Polyacrylamide Gel ,Partial Thromboplastin Time ,Cell Division ,Spleen ,DNA ,Research Article - Abstract
We have designed and synthesized mixed backbone oligonucleotides (MBOs) containing 2'-5'-ribo- and 3'-5'-deoxyribonucleotide segments. Thermal melting studies of the phosphodiester MBOs (three 2'-5'linkages at each end) with the complementary 3'-5'-DNA and -RNA target strands suggest that 2'-5'-ribonucleoside incorporation into 3'-5'-oligodeoxyribonucleotides reduces binding to the target strands compared with an all 3'-5'-oligodeoxyribonucleotide of the same sequence and length. Increasing the number of 2'-5'linkages (from six to nine) further reduces binding to the DNA target strand more than the RNA target strand [Kandimalla,E.R. and Agrawal,S. (1996)Nucleic Acids Symp. Ser., 35, 125-126]. Phosphorothioate (PS) analogs of MBOs destabilize the duplex with the DNA target strand more than the duplex with the RNA target strand. Circular dichroism studies indicate that the duplexes of MBOs with the DNA and RNA target strands have spectral characteristics of both A- and B-type conformations. Compared with the control oligonucleotide, MBOs exhibit moderately higher stability against snake venom phosphodiesterase, S1 nuclease and in fetal calf serum. Although 2'-5'modification does not evoke RNase H activity, this modification does not effect the RNase H activation property of the 3'-5'-deoxyribonucleotide segment adjacent to the modification. In vitro studies with MBOs suggest that they have lesser effects on cell proliferation, clotting prolongation and hemolytic complement lysis than do control PS oligodeoxyribonucleotides. PS analogs of MBOs show HIV-1 inhibition comparable with that of a control PS oligodeoxyribonucleotide with all 3'-5'linkages. The current results suggest that a limited number of 2'-5'linkages could be used in conjunction with PS oligonucleotides to further modulate the properties of antisense oligonucleotides as therapeutic agents.
- Published
- 1997
5. Clinical isolates of HIV-1 contain few pre-existing proteinase inhibitor resistance-conferring mutations
- Author
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Koushi Yamaguchi and Randal A. Byrn
- Subjects
Proteinase inhibitor ,Molecular Sequence Data ,Biophysics ,Human immunodeficiency virus (HIV) ,Biology ,Proteinase Gene ,medicine.disease_cause ,Polymerase Chain Reaction ,Biochemistry ,HIV Protease ,Structural Biology ,Proteinase 3 ,HIV Seropositivity ,medicine ,Humans ,Amino Acid Sequence ,Molecular Biology ,Sequence (medicine) ,Genetics ,Mutation ,Base Sequence ,Sequence Homology, Amino Acid ,Nucleic acid sequence ,Drug Resistance, Microbial ,HIV Protease Inhibitors ,Sequence Analysis, DNA ,Virology ,In vitro ,HIV-1 - Abstract
Proteinase inhibitors are an important new class of antiviral agents for AIDS, however, in vitro experiments have identified proteinase mutations that confer resistance to several different families of the inhibitors. This study was undertaken to determine if these resistance-conferring amino-acid substitutions occur in HIV strains before the application of selective pressure. We determined the nucleic acid sequence of the proteinase gene from the 23 clinical isolates of HIV-1 and three laboratory-adapted strains using a method that detects the majority species present in viral populations. Analysis of minor subpopulations will require alternative strategies. The clinical isolates studied contained an average of 3 (range 1–8_amino-acid substitutions as compared to the prototypical BH10 sequence. We did not detect substitutions characteristic of reported highly proteinase-resistant strains. These results suggest significant variation occurs in the HIV-1 proteinase gene but pre-existing highly proteinase-resistant strains are uncommon.
- Published
- 1995
6. HIV Type 1 Protease Activation of NF-κB within T Lymphoid Cells
- Author
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Nan Zhang, Michael M. Wick, Randal A. Byrn, and Dezhen Zhang
- Subjects
T-Lymphocytes ,medicine.medical_treatment ,Molecular Sequence Data ,Immunology ,Biology ,chemistry.chemical_compound ,HIV Protease ,Transcription (biology) ,Virology ,Gene expression ,medicine ,Humans ,Nuclear protein ,Gene ,Cells, Cultured ,HIV Long Terminal Repeat ,Protease ,Base Sequence ,Oncogene ,NF-kappa B ,Receptors, Interleukin-2 ,NF-κB ,Blotting, Northern ,Molecular biology ,NS2-3 protease ,Infectious Diseases ,chemistry ,Interleukin-2 ,DNA Probes - Abstract
NF-kappa B is a nuclear protein of the rel oncogene family capable of enhancing transcription of several cellular genes, including IL-2 and the IL-2 receptor, and viral genes transcribed from the HIV-1 LTR. It has been reported that HIV-1 protease may cleave the NF-kappa B precursor to its active form in vitro. In this study the effects of HIV protease on NF-kappa B precursor activation were examined in Jurkat T cells by introducing a protease expression vector into the cells. Increased NF-kappa B activity was observed and this increased activity was blocked by a specific inhibitor of the viral protease. Viral transcription, as measured using LTR-CAT assays, was only slightly enhanced in the HIV-protease expressing cells, while secretion of IL-2 and expression of the IL-2 receptor were not affected. The limited activation of NF-kappa B by HIV protease appears unlikely to have a significant effect on virus expression or T cell function.
- Published
- 1995
7. Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides
- Author
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Christopher Lathan, Ekambar R. Kandimalla, Randal A. Byrn, Adrienne Manning, and Sudhir Agrawal
- Subjects
Hot Temperature ,Base Sequence ,Oligonucleotide ,Molecular Sequence Data ,Ribonuclease H ,Nucleic Acid Hybridization ,RNA ,Oligonucleotides, Antisense ,Biology ,Nucleic Acid Denaturation ,Virus Replication ,Antiviral Agents ,Molecular biology ,Structure-Activity Relationship ,Nucleic acid thermodynamics ,Duplex (building) ,HIV-1 ,Genetics ,biology.protein ,Biophysics ,RNA, Viral ,Structure–activity relationship ,Binding site ,RNase H ,Ternary complex - Abstract
Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable ternary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21mer, and evoke RNase H activity. HIV-1 inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases.
- Published
- 1995
8. Corrigendum to 'Isosteric replacements of the carboxylic acid of drug candidate VX-787: Effect of charge on antiviral potency and kinase activity of azaindole-based influenza PB2 inhibitors' [Bioorg. Med. Chem. Lett. 25 (2015) 1990–1994]
- Author
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Mark W. Ledeboer, Michael P. Clark, Tiansheng Wang, Joshua R. Leeman, Randal R. Byrn, Youssef L. Bennani, Hamilton B. Bennett, Ioana Davies, Michael J. Boyd, Wenxin Gu, Upul K. Bandarage, Brian Ledford, Emanuele Perola, Marc Jacobs, and Paul S. Charifson
- Subjects
chemistry.chemical_classification ,Drug candidate ,Stereochemistry ,Carboxylic acid ,Organic Chemistry ,Clinical Biochemistry ,Pharmaceutical Science ,01 natural sciences ,Biochemistry ,Combinatorial chemistry ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,03 medical and health sciences ,0302 clinical medicine ,chemistry ,030220 oncology & carcinogenesis ,Drug Discovery ,Molecular Medicine ,Potency ,Kinase activity ,Molecular Biology - Published
- 2016
9. Targeting of human immunodeficiency virus-infected cells by CD8+ T lymphocytes armed with universal T-cell receptors
- Author
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Lu Qin, Mitchell H. Finer, Annie Chen Tran, Jerome E. Groopman, Daniel J. Capon, Thomas Dull, Margo R. Roberts, Dezhen Zhang, Randal A. Byrn, and Douglas H. Smith
- Subjects
biology ,medicine.medical_treatment ,Immunology ,T-cell receptor ,Cell Biology ,Hematology ,Immunotherapy ,Biochemistry ,Virology ,Cytolysis ,Antigen ,Immunity ,medicine ,biology.protein ,Antibody ,Receptor ,CD8 - Abstract
We have developed an immunotherapeutic approach with potential application in the treatment of viral and malignant disease. We show that primary CD8+ T cells isolated from peripheral blood can be genetically modified by retroviral transduction to express high levels of universal (major histocompatibility complex-unrestricted) chimeric T- cell receptors specific for human immunodeficiency virus (HIV) antigens. Two classes of HIV-specific URs in which the antigen-binding domain is comprised of either CD4 or a single-chain antibody are capable of activating a number of T-cell effector functions in response to target cells, including cytolysis, in a highly sensitive and specific manner. Importantly, we have addressed a number of issues which, although particularly relevant to the clinical application of this approach in the treatment of HIV infection, may also impact on the potential of UR immunotherapy for other disease targets. The UR immunotherapeutic system is particularly suited for evaluation in the clinical setting.
- Published
- 1994
10. Stimulation of Human Immunodeficiency Virus Type 1 Expression by Ceramide
- Author
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Jerome E. Groopman, Randal A. Byrn, Béla Papp, and Dezhen Zhang
- Subjects
Chloramphenicol O-Acetyltransferase ,Ceramide ,Tumor Necrosis Factor-alpha ,Immunology ,Viral transformation ,Lipid signaling ,Transfection ,Biology ,Ceramides ,Immunohistochemistry ,Virology ,Molecular biology ,Virus ,Long terminal repeat ,Chloramphenicol acetyltransferase ,chemistry.chemical_compound ,Infectious Diseases ,chemistry ,HIV-1 ,Humans ,Signal transduction ,Cell Line, Transformed ,HIV Long Terminal Repeat - Abstract
Ceramide, an intracellular lipid mediator of tumor necrosis factor alpha (TNF-alpha) action, was studied for its effects on the expression of the proviral human immunodeficiency virus type 1 genome in latently infected myelomonocytic cell lines U-1IIIB and OM-10.1. Ceramide treatment resulted in a 20- to 100-fold enhancement of HIV production in these cells. Ceramide also enhanced the expression of the chloramphenicol acetyltransferase gene directed by a human immunodeficiency virus type 1 long terminal repeat in transfected U-937 cells, indicating that ceramide acts at the level of viral transcription. These observations suggest that the TNF-ceramide signaling system may be involved in the regulation of HIV expression in certain myeloid cell types.
- Published
- 1994
11. Comparison of the immune response to recombinant gp120 in humans and chimpanzees
- Author
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Phillip W. Berman, David A. Schwartz, Randal A. Byrn, Gerald R. Nakamura, Timothy J. Gregory, Donna J. Eastman, Robert B. Belshe, Geoffrey J. Gorse, Denise M. Wilkes, and Mary Lou Clements
- Subjects
Male ,Pan troglodytes ,viruses ,Molecular Sequence Data ,Immunology ,Antibody Affinity ,Cross Reactions ,HIV Antibodies ,HIV Envelope Protein gp120 ,Protein Structure, Secondary ,Neutralization ,Virus ,Serology ,Epitopes ,Species Specificity ,Antigen ,Neutralization Tests ,Animals ,Humans ,Immunology and Allergy ,Avidity ,Amino Acid Sequence ,AIDS Vaccines ,biology ,Immunogenicity ,Vaccination ,virus diseases ,Virology ,Peptide Fragments ,Recombinant Proteins ,Titer ,Infectious Diseases ,HIV-1 ,biology.protein ,Female ,Antibody ,Protein Binding - Abstract
OBJECTIVE To assess similarities and differences in antibody responses to recombinant (r) HIV-1IIIB gp120 in chimpanzees, previously protected from HIV-1 infection, and human volunteers immunized in connection with a Phase I clinical trial. METHODS Frozen sera from humans immunized with rgp120 from HIV-1IIIB and chimpanzees immunized with the same antigen or recombinant soluble gp160 were compared in a variety of serologic assays. RESULTS The magnitude of the antibody response to gp120 was similar in both species; however, the half-life of the antibody response to rgp120 was approximately 4.5 times longer in humans (9 weeks) than in chimpanzees (2 weeks). Antibodies to gp120 in both species were broadly cross-reactive with gp120 from diverse isolates of HIV-1 and were effective in blocking the binding of gp120 to CD4. Antibody binding to native gp120 was greater than to denatured gp120 in both species. Antibody responses to the principal neutralizing determinant (V3 domain) and virus neutralization titers were approximately 10-fold lower in humans than chimpanzees. The relative avidity of antibody binding to gp120 was higher in the sera from the immunized chimpanzees than in the immunized humans. CONCLUSIONS While the antibody responses to rgp120 elicited in man and chimpanzees were in many ways similar, significant differences did occur. Predictions made on the basis of chimpanzee immunogenicity studies over-estimated the potency of the virus neutralizing titers and under-estimated the duration of the antibody response achieved in humans.
- Published
- 1994
12. Design, structure activity and x-ray crystallographic studies of pseudosymmetrical nonpeptidyl HIV-1 protease inhibitors
- Author
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Robert E. Babine, Nan Zhang, Schow Steven R, Michael M. Wick, Martin F. Semmelhack, Richard C. Hastings, Zhangbao Xu, Suresh S. Kerwar, and Randal A. Byrn
- Subjects
chemistry.chemical_classification ,Protease ,biology ,Chemistry ,Crystal chemistry ,Stereochemistry ,medicine.medical_treatment ,Organic Chemistry ,Clinical Biochemistry ,Pharmaceutical Science ,A protein ,Context (language use) ,Biochemistry ,Chemical synthesis ,Enzyme ,HIV-1 protease ,Enzyme inhibitor ,Drug Discovery ,biology.protein ,medicine ,Molecular Medicine ,Molecular Biology - Abstract
A new class of nonamino acid derived HIV-1 protease inhibitors of structure 1 are described. Structure activity relationships are discussed in the context of a protein crystal complex.
- Published
- 1994
13. Strain specificity and binding affinity requirements of neutralizing monoclonal antibodies to the C4 domain of gp120 from human immunodeficiency virus type 1
- Author
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J A Fox, Brian M. Fendly, M A Norcross, Richard C. Hastings, M R Hobbs, Gerald R. Nakamura, Randal A. Byrn, Phillip W. Berman, D M Wilkes, and H C Wessling
- Subjects
Antigenicity ,medicine.drug_class ,viruses ,Molecular Sequence Data ,Immunology ,Protein domain ,Cross Reactions ,HIV Envelope Protein gp120 ,Monoclonal antibody ,Polymerase Chain Reaction ,Microbiology ,Protein Structure, Secondary ,Epitope ,Cell Line ,Epitopes ,Species Specificity ,Neutralization Tests ,Virology ,medicine ,Humans ,Amino Acid Sequence ,Disulfides ,Binding site ,Binding Sites ,Polymorphism, Genetic ,biology ,Antibodies, Monoclonal ,virus diseases ,Molecular biology ,Primary and secondary antibodies ,Models, Structural ,Kinetics ,Epitope mapping ,Insect Science ,CD4 Antigens ,HIV-1 ,Mutagenesis, Site-Directed ,biology.protein ,Binding Sites, Antibody ,Antibody ,Research Article - Abstract
The binding properties of seven CD4-blocking monoclonal antibodies raised against recombinant gp120 of human immunodeficiency virus type 1 strain MN (HIV-1MN) and two CD4-blocking monoclonal antibodies to recombinant envelope glycoproteins gp120 and gp160 of substrain IIIB of HIVLAI were analyzed. With a panel of recombinant gp120s from seven diverse HIV-1 isolates, eight of the nine antibodies were found to be strain specific and one was broadly cross-reactive. Epitope mapping revealed that all nine antibodies bound to epitopes located in the fourth conserved domain (C4) of gp120. Within this region, three distinct epitopes could be identified: two were polymorphic between HIV-1 strains, and one was highly conserved. Studies with synthetic peptides demonstrated that the conserved epitope, recognized by antibody 13H8, was located between residues 431 and 439. Site-directed mutagenesis of gp120 demonstrated that residue 429 and/or 432 was critical for the binding of the seven antibodies to gp120 from HIV-1MN. Similarly, residues 423 and 429 were essential for the binding of monoclonal antibody 5C2 raised against gp120 from HIV-1IIIB. The amino acids located at positions 423 and 429 were found to vary between strains of HIV-1 as well as between molecular clones derived from the MN and LAI isolates of HIV-1. Polymorphism at these positions prevented the binding of virus-neutralizing monoclonal antibodies and raised the possibility that HIV-1 neutralization serotypes may be defined on the basis of C4 domain sequences. Analysis of the binding characteristics of the CD4-blocking antibodies demonstrated that their virus-neutralizing activity was directly proportional to their gp120-binding affinity. These studies account for the strain specificity of antibodies to the C4 domain of gp120 and demonstrate for the first time that antibodies to this region can be as effective as those directed to the principal neutralizing determinant (V3 domain) in neutralizing HIV-1 infectivity.
- Published
- 1993
14. Structure activity studies on pseudo-symmetrical HIV-1 protease inhibitors
- Author
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Schow Steven R, Suresh S. Kerwar, Michael M. Wick, Robert E. Babine, Randal A. Byrn, Bernard Dean Johnson, Michael R. Jirousek, Parimal R. Desai, Richard C. Hastings, and Nan Zhang
- Subjects
chemistry.chemical_classification ,biology ,Chemistry ,Stereochemistry ,Organic Chemistry ,Clinical Biochemistry ,Human immunodeficiency virus (HIV) ,Pharmaceutical Science ,medicine.disease_cause ,Biochemistry ,chemistry.chemical_compound ,Enzyme ,HIV-1 protease ,Enzyme inhibitor ,Drug Discovery ,biology.protein ,medicine ,Molecular Medicine ,Molecular Biology ,Lead compound - Abstract
Lead compound 1, obtained from a previously reported structure-assisted design approach, was optimized to 17 using a traditional medicinal chemistry approach.
- Published
- 1993
15. Synthesis and biological evaluation of a series of HIV-1 protease inhibitors
- Author
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Randal A. Byrn, Wellington T. Casscles, Michael M. Wick, Robert E. Babine, Schow Steven R, Nan Zhang, Grace C. Hsu, Bernard Dean Johnson, Michael R. Jirousek, Allan Wissner, Richard C. Hastings, Michael P. Trova, and Suresh S. Kerwar
- Subjects
chemistry.chemical_classification ,Protease ,biology ,Chemistry ,medicine.medical_treatment ,Organic Chemistry ,Clinical Biochemistry ,Human immunodeficiency virus (HIV) ,Pharmaceutical Science ,medicine.disease_cause ,Biochemistry ,Virology ,Virus ,In vitro ,Enzyme ,HIV-1 protease ,Enzyme inhibitor ,Drug Discovery ,medicine ,biology.protein ,Molecular Medicine ,Molecular Biology ,Biological evaluation - Abstract
A series of HIV-1 protease inhibitors was prepared and evaluated against the free enzyme for inhibition properties, and for their anti-viral properties in human T lymphoid cells infected with HIVIIIB. Compounds 12, and 19 are the most potent anti-viral agents prepared in this study and are compared to Ro 31-8959, a compound currently in clinical trials for the treatment of AIDS.
- Published
- 1993
16. Monoclonal Antibodies to the Extracellular Domain of HIV-1IIIB gp160 that Neutralize Infectivity, Block Binding to CD4, and React with Diverse Isolates
- Author
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Phillip W. Berman, James P. Porter, Brian M. Fendly, Kim Rosenthal, Maurine R. Hobbs, Donna J. Eastman, Tim Gregory, Gerald R. Nakamura, Lavon Riddle, Donald Dowbenko, and Randal A. Byrn
- Subjects
Protein Conformation ,medicine.drug_class ,viruses ,Immunology ,CHO Cells ,Cross Reactions ,HIV Envelope Protein gp120 ,Monoclonal antibody ,medicine.disease_cause ,Cross-reactivity ,Epitope ,HIV Envelope Protein gp160 ,law.invention ,Epitopes ,Structure-Activity Relationship ,Species Specificity ,Antibody Specificity ,Neutralization Tests ,law ,Cricetinae ,Virology ,Blocking antibody ,medicine ,Animals ,Protein Precursors ,Infectivity ,chemistry.chemical_classification ,biology ,Antibodies, Monoclonal ,Gene Products, env ,Genetic Variation ,virus diseases ,Molecular biology ,Recombinant Proteins ,Infectious Diseases ,chemistry ,CD4 Antigens ,biology.protein ,Recombinant DNA ,Antibody ,Glycoprotein - Abstract
Ten monoclonal antibodies prepared against a soluble, recombinant form of gp160, derived from the IIIB isolate of HIV-1, were characterized. Four of the antibodies neutralized HIV-1IIIB infectivity in vitro, three blocked the binding of recombinant gp120 to CD4, three were reactive with gp41, and one preferentially reacted with an epitope on gp120 within the gp160 precursor. All three CD4 blocking antibodies bound to distinct epitopes, with one mapping to the C1 domain, one mapping to the C4 domain, and one reactive with a conformation-dependent, discontinuous epitope. Of these, the antibody reactive with the discontinuous epitope exhibited neutralizing activity against homologous and heterologous strains of HIV-1. The binding of these monoclonal antibodies to a panel of seven recombinant gp120s prepared from diverse isolates of HIV-1 was measured, and monoclonal antibodies with broad cross reactivity were identified. The epitopes recognized by 7 of the 10 monoclonal antibodies studied were localized by their reactivity with synthetic peptides and with fragments of gp120 expressed as fusion proteins in a lambda gt-11 gp160 epitope library.
- Published
- 1992
17. Conjugation of soluble CD4 without loss of biological activity via a novel carbohydrate-directed cross-linking reagent
- Author
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Randal A. Byrn, Timothy P. Kogan, David H. Peers, Steven M. Chamow, Avi Ashkenazi, and Richard C. Hastings
- Subjects
chemistry.chemical_classification ,Glycoconjugate ,Fluorescence spectrometry ,Biological activity ,Cell Biology ,Hydrazide ,Biochemistry ,Sialic acid ,chemistry.chemical_compound ,chemistry ,Reagent ,Molecular Biology ,Maleimide ,Conjugate - Abstract
Chemical conjugates of recombinant soluble CD4 (sCD4) with toxins, or with antibodies that activate cytotoxic T cells, can be used to direct selective killing of human immunodeficiency virus (HIV)-infected cells. This approach takes advantage of the ability of sCD4 to bind with high affinity to gp120, the envelope protein of HIV-1, which is expressed on actively infected cells. However, conjugation of sCD4 via reagents that target amino groups may reduce its affinity for gp120, since at least one such group is important for gp120 binding. Here, we describe a novel cross-linking reagent which enables the conjugation of sCD4 via its carbohydrate moieties rather than its free amino groups. This heterobifunctional reagent, 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), combines a nucleophilic hydrazide with an electrophilic maleimide, thereby allowing coupling of carbohydrate-derived aldehydes to free thiols. We describe conditions by which MPBH is coupled selectively to the sialic acid residues of sCD4, and exemplify the use of MPBH by conjugating sCD4 to hemoglobin and to beta-galactosidase. We show that, whereas conjugation of sCD4 via amino groups markedly reduces its gp120 binding affinity, conjugation via the carbohydrate chains using MPBH does not affect binding. Moreover, we demonstrate the ability of a sCD4-MPBH-fluorescein conjugate to label HIV-infected human CEM cells selectively. These results indicate that, by targeting its carbohydrate moieties, sCD4 can be cross-linked to other molecules without compromising its function. The approach described here can be useful for glycoproteins in which amino groups, but not carbohydrates, are important for function. More generally, this approach can be considered for use in cross-linking glycoconjugates to compounds which either contain thiols, or to which thiols can be added.
- Published
- 1992
18. Enzymic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments
- Author
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Daniel J. Capon, Randal A. Byrn, Wen Ching Wang, Michael G. Mulkerrin, David H. Peers, Reed J. Harris, Avi Ashkenazi, Steven M. Chamow, and Pamela J. Bjorkman
- Subjects
CD4 immunoadhesin ,Circular dichroism ,Protein Conformation ,Stereochemistry ,Molecular Sequence Data ,CD4 Immunoadhesins ,Plasma protein binding ,HIV Envelope Protein gp120 ,Biology ,Biochemistry ,Receptors, HIV ,Protein structure ,X-Ray Diffraction ,Antigens, CD ,Papain ,Humans ,Amino Acid Sequence ,Protein secondary structure ,Peptide sequence ,Circular Dichroism ,HIV ,Envelope glycoprotein GP120 ,Peptide Fragments ,Antibodies, Anti-Idiotypic ,Immunoglobulin G ,CD4 Antigens ,biology.protein ,Crystallization ,Protein Binding - Abstract
CD4, the cell-surface receptor for the human immunodeficiency virus (HIV), is a member of the immunoglobulin (Ig) gene superfamily. It contains four extracellular sequences homologous to Ig V_L, domains. The first of these (V_1) is sufficient for binding to HIV; however, the structural basis for this binding has yet to be elucidated. While several models for the structure of Ig-like domains in CD4 have been proposed on the basis of crystal structures of Ig V_1, domains, direct evidence that CD4 and VL domains fold similarly has not been obtained. To produce individual domains of CD4 for structural studies, we used molecular fusions of such domains with Ig heavy chain (CD4 immunoadhesins), which are very efficiently expressed and secreted in mammalian cells and can be easily isolated in single-step purification with protein A. Since these fusion molecules are antibody-like homodimeric proteins, we investigated the possibility that they might be cleaved enzymatically to produce Fd-like and Fc fragments. We found that cleavage with papain releases an Fd-like fragment containing the V_1 and V_2 CD4 domains: this fragment fully retains the ability to bind to the HIV-1 envelope glycoprotein gp120 and to block HIV infection in vitro. Moreover, folding of the CD4 domains in the Fd-like fragment and in the parent immunoadhesin is indistinguishable, as indicated by circular dichroism. Spectral analysis of the Fd-like fragment suggests that secondary structure content is identical with that predicted from the known structure of Ig V_L domains; this directly supports the hypothesis that the V_1 and V_2 domains of CD4 fold similarly to Ig V_L domains. Crystals of the Fd-like fragment diffract beyond 3-A resolution and are suitable for detailed structural analysis. The approach described here may be useful, as an alternative to direct expression, in the study of receptors and other adhesion molecules which are members of the Ig gene superfamily.
- Published
- 1990
19. Biological properties of a CD4 immunoadhesin
- Author
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Scot A. Marsters, Jerome E. Groopman, Paul A. Cossum, Steven M. Chamow, Douglas H. Smith, Randal A. Byrn, Catherine Lucas, Timothy J. Gregory, Jennifer S. Johnson, Joyce Mordenti, Florian M. Wurm, and Daniel J. Capon
- Subjects
CD4-Positive T-Lymphocytes ,CD4 immunoadhesin ,CD4 Immunoadhesins ,HIV Envelope Protein gp120 ,Immunoglobulin E ,Virus ,law.invention ,Antigen ,Antigens, CD ,Pregnancy ,law ,Animals ,Humans ,Antibody-dependent cell-mediated cytotoxicity ,Acquired Immunodeficiency Syndrome ,Multidisciplinary ,biology ,Antibody-Dependent Cell Cytotoxicity ,HIV ,virus diseases ,Macaca mulatta ,Virology ,Recombinant Proteins ,Antibodies, Anti-Idiotypic ,Immunoglobulin G ,biology.protein ,Recombinant DNA ,Female ,Antibody ,Immunity, Maternally-Acquired - Abstract
Molecular fusions of CD4, the receptor for human immunodeficiency virus (HIV), with immunoglobulin (termed CD4 immunoadhesins) possess both the gp120-binding and HIV-blocking properties of recombinant soluble CD4, and certain properties of IgG, notably long plasma half-life and Fc receptor binding. Here we show that a CD4 immunoadhesin can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) towards HIV-infected cells, although, unlike natural anti-gp120 antibodies, it does not allow ADCC towards uninfected CD4-expressing cells that have bound soluble gp120 to the CD4 on their surface. In addition, CD4 immunoadhesin, like natural IgG molecules, is efficiently transferred across the placenta of a primate. These observations have implications for the therapeutic application of CD4 immunoadhesins, particularly in the area of perinatal transmission of HIV infection.
- Published
- 1990
20. Effects of Recombinant Soluble CD4 (rCD4) on HIV-1 Infection of Monocyte/Macrophages
- Author
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Paula Pinkston, Scott M. Hammer, Mary Alice Harbison, Richard M. Rose, Jacqueline Gillis, and Randal A. Byrn
- Subjects
U937 cell ,Macrophages ,Microgram ,Monocyte ,Biology ,Virology ,Monocytes ,Recombinant Proteins ,Virus ,In vitro ,Cell Line ,law.invention ,Infectious Diseases ,medicine.anatomical_structure ,Cell culture ,law ,CD4 Antigens ,HIV-1 ,Recombinant DNA ,medicine ,Humans ,Immunology and Allergy ,Macrophage - Abstract
Recombinant soluble CD4 (rCD4) was tested for its ability to block acute human immunodeficiency virus (HIV) infection in the U937 monocytic cell line and in human pulmonary alveolar macrophages (PAM) and for its ability to prevent transfer of virus from chronically infected PAM to target peripheral blood mononuclear leukocytes (PMNL). With an initial virus inoculum of 10(3)-10(4) TCID50/ml, rCD4 completely prevented acute HIV infection of U937 cells at concentrations greater than or equal to 1 microgram/ml and provided substantial but incomplete protection at 0.1 microgram/ml. With an initial virus inoculum of 10(2) TCID50/ml, rCD4 completely prevented acute infection of PAM at concentrations greater than or equal to 0.1 microgram/ml. The transmission of HIV-1 infection to PMNL cocultured with chronically infected PAM was completely inhibited at concentrations greater than or equal to 1 microgram/ml if cell-to-cell contact was prevented. With direct PAM-PMNL contact, substantial inhibition was obtained at an rCD4 concentration of 10 micrograms/ml, and higher concentrations (200 micrograms/ml) could completely block transfer. These results demonstrated that rCD4 can be effective in preventing de novo infection of cells of the monocyte/macrophage lineage, but microenvironments where cell-to-cell contact predominates are likely to pose a formidable challenge to this therapeutic strategy.
- Published
- 1990
21. Synergistic inhibition of HIV-1 by an antisense oligonucleotide and nucleoside analog reverse transcriptase inhibitors
- Author
-
Randal A. Byrn, Sudhir Agrawal, and Gareth J. Veal
- Subjects
endocrine system diseases ,Anti-HIV Agents ,Gene Products, gag ,Pharmacology ,Biology ,Cell Line ,Zidovudine ,chemistry.chemical_compound ,Zalcitabine ,Virology ,medicine ,Humans ,Nucleoside analogue ,Oligonucleotide ,Stavudine ,Drug Synergism ,Oligonucleotides, Antisense ,Thionucleotides ,Dideoxynucleosides ,Mechanism of action ,Biochemistry ,chemistry ,HIV-1 ,Reverse Transcriptase Inhibitors ,Adsorption ,medicine.symptom ,Thymidine ,Nucleoside ,medicine.drug - Abstract
We have studied the effects of the gag antisense phosphorothioate oligonucleotide GEM 91® and mismatch antisense controls on the antiviral activities of ddC and other nucleoside analogs in HIV-infected MT-4 cells using a cytoprotection based assay. Under standard assay conditions, i.e. simultaneous incubation of drugs, HIV-1 IIIB and MT-4 cells, both GEM 91® and mismatch controls interacted synergistically with ddC resulting in an approximate 40-fold decrease in the IC50 value of ddC; this suggests a potent but sequence non-specific effect of GEM 91®. Under post-adsorption assay conditions, i.e. pre-incubation of virus and cells and removal of excess HIV before drug addition, GEM 91® exhibited synergism with ddC, with an approximate 5-fold decrease in ddC IC50 value. This favorable interaction was not seen with any of the mismatch oligonucleotides, suggesting the involvement of a sequence-specific mechanism of action. Similar results were seen with the thymidine analogs AZT and d4T in combination with GEM 91®. These data suggest a potential role for GEM 91® and future sequence-specific antisense drugs in combination with nucleoside analogs for the treatment of HIV infection. It is essential that potential interactions between new and existing classes of anti-HIV drugs are studied extensively as antiretroviral drug combinations become increasingly more complex.
- Published
- 1998
22. Stimulation of HIV expression by intracellular calcium pump inhibition
- Author
-
Randal A. Byrn and Béla Papp
- Subjects
Chloramphenicol O-Acetyltransferase ,Thapsigargin ,Indoles ,medicine.drug_class ,viruses ,chemistry.chemical_element ,Fluorescent Antibody Technique ,Calcium channel blocker ,Calcium-Transporting ATPases ,Calcium ,Biology ,Virus Replication ,Biochemistry ,Calcium in biology ,Virus ,Cell Line ,Chloramphenicol acetyltransferase ,chemistry.chemical_compound ,medicine ,Humans ,Molecular Biology ,Reporter gene ,Terpenes ,Cell Biology ,Virology ,Cell biology ,Hydroquinones ,Kinetics ,chemistry ,HIV-1 ,Cyclopiazonic acid - Abstract
We have studied the role of intracellular calcium sequestration on human immunodeficiency virus (HIV) production by latently infected T-lymphocytic cells. Inhibition of the sarco-endoplasmic reticulum-type calcium transport ATPases by thapsigargin or cyclopiazonic acid induced activation of HIV production in the CEM-derived ACH-2 cells. An approximately 50% depletion of the thapsigargin-sensitive calcium pools as measured fluorimetrically of Indo-loaded cells fully activated virus production. Viral activation was manifest by increases in soluble viral core p24 production, increases in cellular immunofluorescent staining for viral antigens, and increased viral transcription as measured by HIV long terminal repeat-directed expression of the chloramphenicol acetyltransferase reporter gene. Virus induction could be blocked in a dose-dependent manner by the calcium channel blocker econazole. Virus production by the Jurkat-derived HIV-1-inducible J1.1 cells was not significantly stimulated by thapsigargin. These data indicate that intracellular calcium pool function is involved in the control of the transcription of proviral HIV in a cell type-specific manner within the T-lymphoid lineage and that ACH-2 cells represent a useful model for the study of calcium dependent activation of the transcription of proviral HIV.
- Published
- 1995
23. The regulation of HIV by retinoic acid correlates with cellular expression of the retinoic acid receptors
- Author
-
Jerome E. Groopman, Randal A. Byrn, and Koushi Yamaguchi
- Subjects
medicine.drug_class ,Receptors, Retinoic Acid ,T-Lymphocytes ,Immunology ,Molecular Sequence Data ,Retinoic acid ,Alpha (ethology) ,Gene Expression ,Tretinoin ,Retinoid X receptor ,Biology ,Virus Replication ,Monocytes ,Cell Line ,chemistry.chemical_compound ,Macrophages, Alveolar ,medicine ,Immunology and Allergy ,Humans ,Retinoid ,Northern blot ,Receptor ,DNA Primers ,Base Sequence ,Stereoisomerism ,Molecular biology ,Retinoic acid receptor ,Infectious Diseases ,chemistry ,Cell culture ,HIV-1 ,RNA, Viral ,Tetradecanoylphorbol Acetate - Abstract
OBJECTIVES To analyze the effect of retinoic acids (RA) on HIV-1 expression and correlate this effect with expression levels of RA receptors (RARs) in T-lymphoid and monocytoid cell lines. DESIGN AND METHODS The effect of all-trans and 9-cis RA on HIV-1 production in T-lymphoid (H9, CEM) and monocytoid (U937,THP-1) cell lines was measured during acute and chronic infection. The expression levels of human RAR alpha (hRAR alpha, receptor for all-trans RA) and the human retinoid-X receptor alpha (hRXR alpha receptor for 9-cis RA) were determined by Northern blot analysis. RESULTS Both all-trans and 9-cis RA inhibited virus replication in HIV-1 IIIB-infected monocytoid cells, in the presence and absence of the co-stimulatory agent phorbol myristate acetate (PMA). The retinoids had weak or no stimulatory effects on HIV production by T-cell lines. HIV production by PMA-stimulated T-cell lines was inhibited by these retinoids. The 9-cis RA was generally more effective than all-trans RA in inhibiting HIV production and in combination generally more effective than the single agents alone. Human RAR alpha was expressed in H9, U937 and THP-1 cells, but almost undetectable in CEM cells. Human RXR alpha was significantly expressed in U937 and THP-1 cells, weakly expressed in H9 cells and not detectable in CEM cells. After stimulation by PMA, RXR alpha expression increased in H9 and U937 cells but not in CEM cells. Human RAR alpha expression was unchanged in H9 and CEM cells, and elevated in U937 cells, after PMA stimulation. CONCLUSION The effect of RA on HIV-1 expression was cell-type-dependent and partially correlated with cellular expression of RARs. Endogenous or exogenously administered RA may have a significant role in HIV regulation.
- Published
- 1994
24. A humanized, bispecific immunoadhesin-antibody that retargets CD3+ effectors to kill HIV-1-infected cells
- Author
-
Richard P. Junghans, Scot A. Marsters, Dezhen Zhang, Avi Ashkenazi, Randal A. Byrn, Steven M. Chamow, Shilpa M. Mhatre, Xiang Y. Tan, and David H. Peers
- Subjects
CD4-Positive T-Lymphocytes ,Cytotoxicity, Immunologic ,CD3 Complex ,CD3 ,Lymphocyte ,Immunology ,Population ,Blotting, Western ,Antibody Affinity ,Cell Separation ,HIV Antibodies ,HIV Envelope Protein gp120 ,Transfection ,Cell Line ,Immune system ,Antibody Specificity ,Antibodies, Bispecific ,medicine ,Humans ,education ,Killer Cells, Lymphokine-Activated ,Antibody-dependent cell-mediated cytotoxicity ,education.field_of_study ,Hybridomas ,biology ,Antibody-Dependent Cell Cytotoxicity ,virus diseases ,Hematology ,Cytotoxicity Tests, Immunologic ,Virology ,Molecular biology ,Recombinant Proteins ,CTL ,medicine.anatomical_structure ,Immunoglobulin G ,CD4 Antigens ,biology.protein ,HIV-1 ,Interleukin-2 ,Electrophoresis, Polyacrylamide Gel ,Antibody ,CD8 ,CD4 Immunoadhesins ,Muromonab-CD3 ,T-Lymphocytes, Cytotoxic - Abstract
We have developed a humanized, bispecific immunoadhesin-antibody (BsIAb) that targets and kills HIV-infected cells. Comprised of CD4-IgG and humanized anti-CD3-IgG, this BsIAb is bifunctional. First, in targeting, it exploits the natural affinity of CD4 for gp120 to target the BsIAb to HIV-infected cells, and second, it recruits and activates, through its anti-CD3 moiety, cytotoxic T lymphocytes (CTL) to lyse target cells in a non-MHC restricted manner. To produce purified BsIAb from supernantants of transfected mammalian cells, we designed a three-step recovery scheme based on the structural elements of this heterotrimeric protein. The ability of purified BsIAb to specifically lyse HIV-infected target cells was demonstrated using CTL from two different sources: whole peripheral blood lymphocyte (PBL) fractions and pure CTL preparations. In contrast, a human anti-gp120 antibody mediated lysis of HIV-infected target cells only with PBL fractions and not with purified CTL. Moreover, lysis observed in the presence of the human anti-gp120 antibody was completely blocked in the presence of human serum (which competes for Fc gamma receptor binding), whereas BsIAb-mediated lysis of target cells was not affected. We measured the monovalent affinities of BsIAb for HIV-gp120 on infected cells and for CD3 epsilon on CTL. Relative to the bivalent parent molecules, CD4/gp120 affinity in the BsIAb is unchanged, whereas anti-CD3/CD3 is substantially decreased. We further demonstrated by fluorescence microscopy that physical association of CD3+ cells with gp120-expressing cells occurs only in the presence of BsIAb. Thus, the cytocidal activity of BsIAb in the presence of serum reflects its unique ability to recruit CTL as effector cells and highlights a potentially important advantage of this type of construct over antibodies for HIV-directed therapy.
- Published
- 1994
25. Characterization of autoantibodies to the CD4 molecule in human immunodeficiency virus infection
- Author
-
K. Ikeuchi, I. Sekigawa, Randal A. Byrn, Kiyoshi Takatsuki, J. D. Allan, J. E. Groopmen, and G. Biberfield
- Subjects
biology ,medicine.drug_class ,Lymphocyte ,Immunology ,Autoantibody ,HIV Infections ,Monoclonal antibody ,Virology ,Molecular biology ,Virus ,Pathology and Forensic Medicine ,law.invention ,medicine.anatomical_structure ,Antigen ,law ,CD4 Antigens ,biology.protein ,medicine ,Recombinant DNA ,Immunology and Allergy ,Humans ,Antibody ,CD8 ,Autoantibodies - Abstract
Autoantibodies to the CD4 protein, which serves as a receptor for the human immunodeficiency virus (HIV) on the surface of target cells, were found in patients with different stages of HIV disease. Using recombinant soluble CD4 (rCD4) antigen in a enzyme-linked immunosorbent assay (ELISA), we detected serum anti-CD4 antibodies in approximately 20% of HIV-1 infected patients and 13% of HIV-2 infected patients. There was no correlation between the presence of anti-CD4 antibodies and the stage of HIV disease, serum IgG concentration, number of peripheral blood CD4 positive lymphocytes, or CD4 CD8 lymphocyte ratios in HIV-1 infected patients. Immunoaffinity purified anti-CD4 antibody failed to bind to CD4 positive cells using flow cytometric analysis. However, this antibody could weakly bind to CD4 positive cells that had been preincubated with purified recombinant gp120 (rgp120). In addition, using an ELISA system, we found that the binding of purified patient anti-CD4 antibody to rCD4 was increased in the presence of rgp120. Similar increased binding was observed with the anti-CD4 monoclonal antibody OKT4, but not with anti-Leu3a. These data suggest that a conformational change in the C-terminal domains of CD4 may be induced by gp120 binding and could lead to development of anti-CD4 antibodies.
- Published
- 1991
26. Infection of nonlymphoid cells by human immunodeficiency virus type 1 or type 2
- Author
-
Sunyoung Kim, Jerome E. Groopman, Kenji Ikeuchi, Randal A. Byrn, and S R Goldring
- Subjects
Chloramphenicol O-Acetyltransferase ,Mesenchyme ,Immunology ,Transfection ,Microbiology ,Virus ,Cell Line ,Antigen ,Viral entry ,Antigens, CD ,Virology ,medicine ,Humans ,Lymphocytes ,Lung ,Skin ,CD40 ,biology ,Mesenchymal stem cell ,Cell Transformation, Viral ,Molecular biology ,Recombinant Proteins ,Kinetics ,medicine.anatomical_structure ,Cartilage ,Cell culture ,Insect Science ,CD4 Antigens ,DNA, Viral ,HIV-2 ,biology.protein ,HIV-1 ,Research Article ,Plasmids - Abstract
Human epithelial cells (L132) derived from embryonic lung and human lung fibroblasts (MRC5) were infected by human immunodeficiency virus type 1 (HIV-1) or type 2 (HIV-2). Surface CD4 protein was detected on these cells, and recombinant soluble CD4 (sCD4) blocked infection, indicating that HIV infection was mediated by the cell surface CD4 protein. In contrast, infection of human primary chondrocyte cells (C23), synovial cells (HSA), and foreskin fibroblasts (F13) was apparently independent of cell CD4-mediated mechanisms. Surface CD4 protein could not be detected on these cells, and sCD4 did not block the infection. F13 cells could be infected only by HIV-2, not by HIV-1, under our experimental conditions. In cells of mesenchymal orgin, viral production could be detected only after cocultivation with the human T-lymphoid H9 cells but not by conventional viral assays, including reverse transcriptase and p24 antigen assays in cell culture supernatant and immunofluorescence of host cells. Our DNA transfection studies indicated that this lack of detectable viral production was not due to the inefficient use of the HIV long terminal repeat or the Tat protein in these cells. These mesenchymal and epithelial cells were susceptible to HIV infection but differed in mechanism of virus entry compared with hematopoietic cells such as T lymphocytes. These observations may provide insights into clinical syndromes such as lung dysfunction in HIV-infected newborns and connective tissue disorders in HIV-infected adults.
- Published
- 1990
27. Expression and immunogenicity of the extracellular domain of the human immunodeficiency virus type 1 envelope glycoprotein, gp160
- Author
-
O K Haffar, Lavon Riddle, Phillip W. Berman, Gerald R. Nakamura, P Skehel, Randal A. Byrn, Tim Gregory, Wylla M. Nunes, Jerome E. Groopman, and T J Matthews
- Subjects
Antigens, Differentiation, T-Lymphocyte ,HIV Antigens ,viruses ,Immunology ,Retroviridae Proteins ,Mutagenesis (molecular biology technique) ,HIV Envelope Protein gp120 ,Biology ,Gp41 ,Binding, Competitive ,Microbiology ,Chromatography, Affinity ,Virus ,Cell Line ,HIV Envelope Protein gp160 ,Viral Envelope Proteins ,Viral envelope ,Affinity chromatography ,Virology ,Protein purification ,Animals ,Humans ,chemistry.chemical_classification ,virus diseases ,Precipitin Tests ,Molecular biology ,Amino acid ,Gene Expression Regulation ,chemistry ,Insect Science ,Mutation ,HIV-1 ,Glycoprotein ,Plasmids ,Research Article - Abstract
The envelope glycoprotein of human immunodeficiency virus type 1 is synthesized as a precursor, gp160, that subsequently is cleaved to yield mature gp120 and gp41. In these studies, the gene encoding gp160 was mutagenized so as direct the synthesis of a truncated protein consisting of the extracellular domains of both gp120 and gp41. The variant protein, termed sgp160, consisted of 458 amino acids of gp120 and 172 amino acids of gp41. To facilitate protein purification, the normal polyglycoprotein processing site between gp120 and gp41 was deleted through the use of site-directed mutagenesis. This allowed for the synthesis of a molecule that could be purified by affinity chromatography, using acid elution, without dissociation of the gp120 polypeptide from the gp41 polypeptide. The conformation of the sgp160 variant appeared to be functionally relevant, as reflected by its ability to bind to CD4 with an affinity comparable to that of the variant rgp120. The structure of the sgp160-containing polypeptide differed from that of rgp120 in that it tended to form high-molecular-weight aggregates that could be dissociated to monomers and dimers in the presence of reducing agents. Antibodies against the sgp160 protein reacted with authentic virus-derived gp160, gp120, and gp41; neutralized viral infectivity; and inhibited the binding of rgp120 to CD4. Rabbit antibodies to the sgp160 protein differed from those raised against rgp120 in that they were enriched for populations that blocked CD4 binding but did not prevent human immunodeficiency virus type 1-induced syncytium formation.
- Published
- 1989
28. CD4+ lymphocyte function with early human immunodeficiency virus infection
- Author
-
Kenneth C. Anderson, R J Gurley, Randal A. Byrn, Kenji Ikeuchi, and Jerome E. Groopman
- Subjects
Antigens, Differentiation, T-Lymphocyte ,CD4-Positive T-Lymphocytes ,Lymphocyte ,Retroviridae Proteins ,AIDS-related complex ,Lymphocyte proliferation ,HIV Envelope Protein gp120 ,Lymphocyte Activation ,Major histocompatibility complex ,Antibodies ,Immune system ,Antigen ,AIDS-Related Complex ,HIV Seropositivity ,Tetanus Toxoid ,medicine ,Humans ,Phytohemagglutinins ,Acquired Immunodeficiency Syndrome ,Multidisciplinary ,biology ,Ionomycin ,Toxoid ,virus diseases ,T-Lymphocytes, Helper-Inducer ,medicine.disease ,Antigens, Differentiation ,Virology ,Recombinant Proteins ,medicine.anatomical_structure ,Immunology ,biology.protein ,Tetradecanoylphorbol Acetate ,Antibody ,Cell Division ,Ethers ,Research Article - Abstract
The pathogenesis of cellular immune deficiency following human immunodeficiency virus (HIV) infection could result from quantitative and/or qualitative dysfunction of the CD4+ lymphocyte population. To better characterize the T-cell response to soluble antigen with HIV infection, we have isolated peripheral blood lymphocytes and purified populations of CD4+ lymphocytes from healthy HIV antibody-positive subjects, patients with acquired immunodeficiency syndrome (AIDS)-related complex (ARC), and healthy HIV antibody-negative controls. T-lymphocyte function was determined by proliferative response to lectin (phytohemagglutinin), phorbol 12-myristate 13-acetate (PMA), calcium ionophore, purified recombinant HIV envelope gp120, tetanus toxoid antigen, and tetanus toxoid antigen in the presence of recombinant gp120 or purified recombinant soluble CD4. PBLs and CD4+ lymphocytes from asymptomatic HIV-infected subjects responded equally well to lectin, PMA, and/or calcium ionophore and to tetanus toxoid as cells from uninfected control subjects. The cells that proliferated in response to a soluble antigenic stimulus did not respond to gp120. Cells from subjects with ARC had a selective antigen recognition defect independent of the number of CD4+ lymphocytes. Recombinant gp120 inhibited CD4+ lymphocyte proliferation to antigenic stimulus by 30-40%. Recombinant soluble CD4, a proposed therapeutic for HIV, had no effect on T-cell response to antigen. A selective antigen recognition response was not compromised early in HIV infection but was compromised in subjects with ARC. Inhibition of proliferation to tetanus toxoid by gp120 suggests that HIV may affect major histocompatibility complex II restricted antigen recognition independent of CD4+ cell loss.
- Published
- 1989
29. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression
- Author
-
Randal A. Byrn, Sunyoung Kim, David Baltimore, and Jerome Groopman
- Subjects
viruses ,Immunology ,HIV ,RNA ,RNA-dependent RNA polymerase ,Biology ,Microbiology ,Virology ,Molecular biology ,Cell Line ,RNA silencing ,Gene Expression Regulation ,Viral replication ,Viral entry ,DNA-directed RNA interference ,Insect Science ,DNA, Viral ,Gene expression ,Viral structural protein ,Humans ,RNA, Viral ,Caltech Library Services ,Research Article - Abstract
The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. We have established a single-cycle growth condition for HIV in H9 cells, a human CD4+ lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes.
- Published
- 1989
30. Designing CD4 immunoadhesins for AIDS therapy
- Author
-
Douglas H. Smith, Scot A. Marsters, Catherine Lucas, Samuel Broder, Joyce Mordenti, Timothy J. Gregory, Steven M. Chamow, Daniel J. Capon, Randal A. Byrn, Hiroaki Mitsuya, Florian M. Wurm, and Jerome E. Groopman
- Subjects
Retroviridae Proteins ,CD4 Immunoadhesins ,Receptors, Fc ,HIV Envelope Protein gp120 ,Immunoglobulin E ,Binding, Competitive ,Virus ,Cell Line ,Mice ,Receptors, HIV ,Viral envelope ,Acquired immunodeficiency syndrome (AIDS) ,Antigen ,Adjuvants, Immunologic ,medicine ,Animals ,Humans ,Acquired Immunodeficiency Syndrome ,Multidisciplinary ,biology ,virus diseases ,medicine.disease ,Virology ,Receptors, Complement ,Drug Design ,Immunoglobulin G ,Immunology ,Antigens, Surface ,biology.protein ,Receptors, Virus ,Viral disease ,Binding Sites, Antibody ,Rabbits ,Antibody ,Cell Adhesion Molecules ,Half-Life - Abstract
A newly-constructed antibody-like molecule containing the gp120-binding domain of the receptor for human immunodeficiency virus blocks HIV-1 infection of T cells and monocytes. Its long plasma half-life, other antibody-like properties, and potential to block all HIV isolates, make it a good candidate for therapeutic use.
- Published
- 1989
31. Blocking of HIV-1 infectivity by a soluble, secreted form of the CD4 antigen
- Author
-
Douglas H. Smith, Randal A. Byrn, Scot A. Marsters, Daniel J. Capon, Timothy J. Gregory, and Jerome E. Groopman
- Subjects
Antigens, Differentiation, T-Lymphocyte ,CD4 antigen ,viruses ,T-Lymphocytes ,Virus ,Cell Line ,chemistry.chemical_compound ,Viral envelope ,Antigen ,Viral Envelope Proteins ,Animals ,Humans ,Infectivity ,Acquired Immunodeficiency Syndrome ,Multidisciplinary ,biology ,virus diseases ,HIV ,Transfection ,Envelope glycoprotein GP120 ,Virology ,Recombinant Proteins ,chemistry ,Cell culture ,biology.protein ,Receptors, Virus - Abstract
The initial event in the infection of human T lymphocytes, macrophages, and other cells by human immunodeficiency virus (HIV-1) is the attachment of the HIV-1 envelope glycoprotein gp120 to its cellular receptor, CD4. As a step toward designing antagonists of this binding event, soluble, secreted forms of CD4 were produced by transfection of mammalian cells with vectors encoding versions of CD4 lacking its transmembrane and cytoplasmic domains. The soluble CD4 so produced binds gp120 with an affinity and specificity comparable to intact CD4 and is capable of neutralizing the infectivity of HIV-1. These studies reveal that the high-affinity CD4-gp120 interaction does not require other cell or viral components and may establish a novel basis for therapeutic intervention in the acquired immune deficiency syndrome (AIDS).
- Published
- 1987
32. Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1
- Author
-
Jerome E. Groopman, Sunyoung Kim, David Baltimore, Randal A. Byrn, and Kenji Ikeuchi
- Subjects
DNA Replication ,Genes, Viral ,viruses ,Genetic Vectors ,Biology ,Virus ,Gene Products, nef ,Cell Line ,Gene product ,Viral entry ,Transcription (biology) ,Gene expression ,Humans ,Viral Regulatory and Accessory Proteins ,nef Gene Products, Human Immunodeficiency Virus ,Gene ,Multidisciplinary ,RNA ,virus diseases ,Virology ,Molecular biology ,Long terminal repeat ,Genes, nef ,Kinetics ,DNA, Viral ,HIV-1 ,RNA, Viral ,Research Article - Abstract
Human immunodeficiency virus type 1 (HIV-1) contains an open reading frame called nef at the 3' end of its genome. The nef gene product has been reported to down-regulate viral growth by suppressing viral transcription through interaction with the long terminal repeat region. We have compared two isogenic HIV-1 (HIV-1-WI3) strains, one of which lacks nef expression, and found little difference between them in in vitro growth. We tested effects on viral entry, DNA synthesis, and RNA expression by measuring HIV-specific low molecular weight DNA and RNA after infection. The qualitative and quantitative aspects of DNA and RNA synthesis were comparable between the nef+ and nef- strains. The effects on viral growth were also examined by following changes in reverse transcriptase activity during the course of infection. The presence of the nef gene product failed to slow viral growth in several different cell types tested, including the human T-lymphocyte cell lines H9 and CEM-SS, human primary T cells enriched for CD4+ cells, and human monocytic cell lines U-937 and THP-1. On the contrary, the nef+ strain grew more efficiently in some cell types than the nef- strain. The same results were obtained with nef+ and nef- strains of a different virus, HIV-1-432, whose Nef had been reported to have a negative effect on viral growth. Our data suggest that the Nef protein does not act as a negative factor, at least in the experimental systems employed in our studies.
- Published
- 1989
33. Production of tumor necrosis factor alpha and interleukin 1 beta by monocytic cells infected with human immunodeficiency virus
- Author
-
David T. Scadden, Jerome E. Groopman, Randal A. Byrn, J M Molina, and Charles A. Dinarello
- Subjects
Lipopolysaccharides ,HIV Antigens ,medicine.medical_treatment ,HIV Core Protein p24 ,Retroviridae Proteins ,Biology ,Monocytes ,Cell Line ,Tissue culture ,medicine ,Tumor Cells, Cultured ,Humans ,Northern blot ,Beta (finance) ,Acquired Immunodeficiency Syndrome ,Tumor Necrosis Factor-alpha ,Monocyte ,Interleukin ,RNA-Directed DNA Polymerase ,General Medicine ,Molecular biology ,Cytokine ,medicine.anatomical_structure ,Cell culture ,Immunology ,Leukemia, Monocytic, Acute ,Tumor necrosis factor alpha ,Interleukin-1 ,Research Article - Abstract
The production of tumor necrosis factor alpha (TNF alpha) and IL-1 beta by the monocytic cell line THP-1, productively infected with HIV-1, was investigated using specific RIA and Northern blot analysis. HIV-infected cells, like uninfected cells, did not constitutively produce any detectable amounts of protein or mRNA for TNF alpha or IL-1 beta. After stimulation with LPS or a combination of LPS plus IFN-gamma, TNF alpha and IL-1 beta were detected in tissue culture supernatants and cell lysates and transcripts for both cytokines were seen on Northern blots. No significant difference in production of these two cytokines was observed between uninfected and chronically infected cells. Acutely HIV-infected cells, however, showed phenotypic changes compatible with maturation and an increase in TNF alpha and IL-1 beta mRNA production, and released significantly higher levels of TNF alpha and IL-1 beta compared with chronically infected or uninfected cells. Furthermore, LPS stimulation of HIV-infected cells increased virus production. These results suggest that HIV-infected monocytic cells may produce increased amounts of TNF alpha and IL-1 beta in response to stimuli that could be present in vivo.
- Published
- 1989
34. Characterization of in vitro inhibition of human immunodeficiency virus by purified recombinant CD4
- Author
-
S M Chamow, Randal A. Byrn, Jerome E. Groopman, I Sekigawa, J S Johnson, T J Gregory, and Daniel J. Capon
- Subjects
Antigens, Differentiation, T-Lymphocyte ,Immunology ,Retroviridae Proteins ,Biology ,HIV Envelope Protein gp120 ,Microbiology ,Virus ,law.invention ,Cell Line ,Receptors, HIV ,Viral envelope ,Viral entry ,law ,Virology ,chemistry.chemical_classification ,virus diseases ,HIV ,Biological activity ,In vitro ,Recombinant Proteins ,chemistry ,Cell culture ,Insect Science ,Antigens, Surface ,Recombinant DNA ,Receptors, Virus ,Adsorption ,Glycoprotein ,Research Article - Abstract
The first step in infection of human T cells with human immunodeficiency virus (HIV) is binding of viral envelope glycoprotein gp120 to its cellular receptor, CD4. The specificity of this interaction has led to the development of soluble recombinant CD4 (rCD4) as a potential antiviral and therapeutic agent. We have previously shown that crude preparations of rCD4 can indeed block infection of T cells by HIV type 1 (HIV-1). Here we present a more detailed analysis of this antiviral activity, using HIV-1 infection of the T lymphoblastoid cell line H9 as a model. Purified preparations of rCD4 blocked infection in this system at nanomolar concentrations; combined with the known affinity of the CD4-gp120 interaction, this finding suggests that the inhibition is simply due to competition for gp120 binding. As predicted, rCD4 had comparable activity against all strains of HIV-1 tested and significant activity against HIV-2. Higher concentrations of rCD4 blocked infection even after the virus had been adsorbed to the cells. These findings imply that the processes of viral adsorption and penetration require different numbers of gp120-CD4 interactions. Recombinant CD4 was able to prevent the spread of HIV infection in mixtures of uninfected and previously infected cells. Our studies support the notion that rCD4 is a potent antiviral agent, effective against a broad range of HIV-1 isolates, and demonstrate the value of purified rCD4 as an experimental tool for studying the mechanism of virus entry into cells.
- Published
- 1989
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