ABSTRACT
The tight junctions of the cerebral capillary endothelium form the highly restrictive blood-brain barrier. Migration of leukocytes across this unique barrier may involve ligation of elements in addition to those of the fenestrated capillaries of the peripheral vascular system. An antibody raised against a bacterial adhesive protein and shown to have cross-reactivity with brain capillaries and to block leukocyte migration into the brain was used to identify and clone a novel determinant on brain microvessels. This cDNA was sequenced, and the expressed protein supported leukocyte adhesion in a CD18-dependent fashion. The high level of brain microvessel expression of this adhesion molecule, termed the cerebral cell adhesion molecule, implicates it in leukocyte transmigration across the blood-brain barrier.
Subject(s)
Blood-Brain Barrier , Capillaries , Cell Adhesion Molecules/immunology , Cerebral Cortex/blood supply , Endothelium, Vascular , Virulence Factors, Bordetella , Adhesins, Bacterial/immunology , Cell Adhesion , Cell Adhesion Molecules/isolation & purification , Hemagglutinins/immunology , Humans , Neutrophils/physiology , Tight Junctions , Tissue DistributionABSTRACT
A phosphorothioate c-myc antisense oligonucleotide was complexed with zinc and encapsulated into injectable biodegradable microspheres. The efficacy of this novel formulation was compared with intravenous administration of the unencapsulated drug in human melanoma and leukemia xenografts in immunocompromised mice. The microencapsulated formulation was more effective as shown by reduced tumor growth, a decreased number of metastases, reduced c-myc expression, and increased survival in the melanoma model, and decreased metastatic potential and increased survival in the leukemia model. These results show that, as has been demonstrated previously with protein and peptide drugs, greater therapeutic efficacy can be obtained when antisense oligonucleotides are delivered from sustained-release formulations.
Subject(s)
Antineoplastic Agents/pharmacology , Genes, myc , Leukemia/drug therapy , Melanoma/drug therapy , Oligonucleotides, Antisense/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Base Sequence , DNA Primers , Humans , Male , Mice , Mice, Nude , Microspheres , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides, Antisense/therapeutic useABSTRACT
To explore the ability to use genetic fusions of transferrin as a carrier for brain targeting and delivery, a series of fusion proteins containing both human nerve growth factor (NGF) and human transferrin was produced in mammalian cells. A protein in which the hinge region from human IgG3 joined the carboxyl terminus of NGF and the amino terminus of transferrin formed a covalent homodimer, bound human transferrin receptor, and retained full NGF in PC12 cells. In contrast, proteins in which polypeptide dimerization was not induced or in which NGF was fused through its amino terminus had greatly reduced NGF activity. The ability to maintain both biologically active NGF and transferrin as part of a fusion protein may offer a novel way to deliver NGF and other neurotrophic factors to the central nervous system.
Subject(s)
Blood-Brain Barrier , Brain/metabolism , Drug Delivery Systems , Nerve Growth Factors/biosynthesis , Nerve Growth Factors/pharmacology , Receptors, Transferrin/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/pharmacology , Transferrin/administration & dosage , Drug Carriers , Enzyme-Linked Immunosorbent Assay , Humans , Polymerase Chain ReactionABSTRACT
Two recent advances have enabled the development of clinically useful, injectable, sustained-release protein formulations. The first is a nonaqueous, cryogenic atomization process that encapsulates the protein into microspheres composed of a biodegradable polymer from which the protein is released slowly. The second consists of numerous ways of maintaining protein stability within the microspheres for extended periods after injection. In addition to allowing less frequent administration of protein drugs, at possibly lower overall doses, it is possible that sustained-release formulations will justify commercial development of proteins that could not be marketed as solution formulations.
Subject(s)
Drug Delivery Systems , Proteins/administration & dosage , Delayed-Action Preparations , Drug Compounding , Drug Stability , Humans , Injections , MicrospheresSubject(s)
Drug Compounding , Human Growth Hormone/chemistry , Microspheres , Animals , Human Growth Hormone/blood , Lactic Acid/chemistry , Macaca mulatta , Male , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Polymers/chemistry , Rats , Rats, Sprague-Dawley , Recombinant Proteins/blood , Recombinant Proteins/chemistryABSTRACT
Although numerous protein therapeutics have been approved or are in advanced clinical testing, the development of more sophisticated delivery systems for this rapidly expanding class of therapeutic agents has not kept pace. The short in vivo half-lives, the physical and chemical instability, and the low oral bioavailability of proteins currently necessitate their administration by frequent injections of protein solutions. This problem can be overcome by use of injectable depot formulations in which the protein is encapsulated in, and released slowly from, microspheres made of biodegradable polymers. Although the first report of sustained release of a microencapsulated protein was more than 20 years ago, the instability of proteins in these dosage forms has prevented their clinical use. Advances in protein stabilization, however, have allowed development of sustained-release forms of several therapeutic proteins, and clinical testing of a monthly formulation human growth hormone is currently in progress. The obvious advantage of this method of delivery is that the protein is administered less frequently, sometimes at lower overall doses, than when formulated as a solution. More importantly, it can justify commercial development of proteins that, for a variety of reasons, could not be marketed as solution formulations.
Subject(s)
Drug Delivery Systems , Proteins/administration & dosage , Proteins/therapeutic use , Animals , Delayed-Action Preparations , HumansABSTRACT
PURPOSE: To produce and evaluate sustained-acting formulations of recombinant human growth hormone (rhGH) made by a novel microencapsulation process. METHODS: The protein was stabilized by forming an insoluble complex with zinc and encapsulated into microspheres of poly (D,L-lactide co-glycolide) (PLGA) which differed in polymer molecular weight (8-31 kD), polymer end group, and zinc content. The encapsulation procedure was cryogenic, non-aqueous, and did not utilize surfactants or emulsification. The rhGH extracted from each of these microsphere formulations was analyzed by size-exclusion, ion-exchange and reversed-phase chromatography, SDS-polyacrylamide gel electrophoresis, peptide mapping, and cell proliferation of a cell line expressing the hGH receptor. In addition, the in vivo release profile was determined after subcutaneous administration of the microspheres to rats and juvenile rhesus monkeys. RESULTS: Protein and bioactivity analyses of the rhGH extracted from three different microsphere formulations showed that the encapsulated protein was unaltered relative to the protein before encapsulation. In vivo, microsphere administration to rats or monkeys induced elevated levels of serum rhGH for up to one month, more than 20-fold longer than was induced by the same amount of protein injected subcutaneously as a solution. The rate of protein release differed between the three microsphere formulations and was determined by the molecular weight and hydrophobicity of the PLGA. The serum rhGH profile, after three sequential monthly doses of the one formulation examined, was reproducible and showed no dose accumulation. CONCLUSIONS: Using a novel process, rhGH can be stabilized and encapsulated in a solid state into PLGA microspheres and released with unaltered properties at different rates.
Subject(s)
Human Growth Hormone/chemistry , Lactic Acid , Polyglycolic Acid , Administration, Cutaneous , Animals , Capsules , Chromatography, Gel , Chromatography, Ion Exchange , Drug Compounding , Drug Stability , Human Growth Hormone/administration & dosage , Human Growth Hormone/blood , Macaca mulatta , Male , Polylactic Acid-Polyglycolic Acid Copolymer , Polymers , Rats , Rats, Sprague-Dawley , Recombinant Proteins/administration & dosage , Recombinant Proteins/blood , Recombinant Proteins/chemistryABSTRACT
Long-acting formulations of recombinant human growth hormone (rhGH) were prepared by stabilizing and encapsulating the protein into three different injectable, biodegradable microsphere formulations composed of polymers of lactic and glycolic acid. The formulations were compared in juvenile rhesus monkeys by measuring the serum levels of rhGH and two proteins induced by hGH, insulin-like growth factor-I and IGF binding protein-3 (IGFBP-3) after single s.c. administration. All three formulations, which differed principally in the composition of the polymer, provided sustained elevated levels of all three proteins for several weeks, and the rate of release of rhGH differed among the formulations consistent with the molecular weight of the polymer used. All three formulations induced a higher level of insulin-like growth factor-I and insulin-like growth factor binding protein than was induced by daily injections of the same amount of rhGH in solution. After three monthly injections of one of the formulations, both the rhGH and IGF-I levels remained elevated for nearly 90 days. Immunogenicity of the rhGH released from this formulation, as assessed by the incidence of seroconversion to hGH and the titer of anti-hGH antibody in both the rhesus monkeys and transgenic mice expressing rhGH, was no greater than that of the unencapsulated protein. In addition, the microsphere injection sites appeared normal by macroscopic evaluation between 1 to 2 mo after microsphere administration and by microscopic evaluation between 2 to 3 mo. These results show that serum levels of a therapeutic protein can be sustained for an extended period when encapsulated into different formulations of injectable, biodegradable microspheres.
Subject(s)
Drug Delivery Systems , Formularies as Topic , Growth Hormone/metabolism , Recombinant Proteins/metabolism , Animals , Humans , Macaca mulatta , Male , Mice , Mice, Inbred BALB C , Time FactorsABSTRACT
The cDNAs encoding the variable regions of the heavy and light chains of a murine antibody specific for the human TfR were cloned and a human chimera (gamma1, kappa) was produced. A gene fusion was created by joining the 3' end of the coding region of the human nerve growth factor (NGF) precursor to the 5' end of the heavy chain variable region of the chimeric antibody. When expressed with the unmodified light chain in mammalian cells, the protein fusion is properly processed, assembled, and secreted. Subsequent purification and characterization established the uncompromised bifunctional activities of the protein, relative to the unmodified components, as demonstrated by its ability to both bind to the human TfR and induce neurite outgrowth in primary sympathetic or spinal ganglia and in trkA-transfected pheochromocytoma cells. The ability to generate biologically active NGF fused to a TfR targeting antibody, which was previously shown to cross the blood-brain barrier, may offer a novel way to deliver NGF and other neurotrophic factors to the central nervous system.
Subject(s)
DNA, Complementary/isolation & purification , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Light Chains/biosynthesis , Nerve Growth Factors/genetics , Receptors, Transferrin/immunology , Animals , Antibody Specificity , Chimera/genetics , Cloning, Molecular , Genetic Code , Humans , Immunoglobulin Variable Region , Mice , Molecular Sequence Data , Nerve Growth Factors/biosynthesis , Recombinant Fusion Proteins/biosynthesisABSTRACT
The development of a sustained release formulation of recombinant human growth hormone (rhGH) has focused on a depot preparation using the biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA), for microsphere production. These formulations have been designed to assure the maintenance of protein integrity both during the microencapsulation process and upon subsequent release in vitro and in vivo. In addition, animal models were developed to assess both the in vivo release kinetics and the potency of the released protein. These studies emphasized the importance of obtaining a correlation between the in vivo and in vitro release at an early stage of development. Juvenile rhesus monkey studies revealed that continuous rhGH administration resulted in a greater total insulin-like growth factor-I (IGF-I) response than daily rhGH administration, indicating that a continuous rhGH dose may provide comparable efficacy to daily dosing at a lower total dose of rhGH. The use of a conventional water-in-oil-in-water process yielded a triphasic release of biologically active and non-immunogenic rhGH, while the novel cryogenic process achieved a continuous release of rhGH that is biologically active and non-immunogenic. The rhGH PLGA formulation produced by the novel cryogenic process was manufactured under aseptic GMP conditions and was shown to be safe in growth hormone-deficient adults. This protein and these studies should serve as a model for the future development of PLGA formulations for therapeutic proteins.
ABSTRACT
Monoclonal antibodies to the human transferrin receptor were screened for binding to capillary vessels in human, monkey, rabbit and rat brain tissue. Two antibodies were selected that bind both human and monkey but not rabbit or rat microvessels. With recombinant fragments of the human receptor, both antibodies were shown to bind to a region of the extracellular portion of the receptor that is relatively variable among species. Binding, which was characterized by using purified receptor and K562 cells, was not reduced by excess transferrin, indicating that the antibodies bind the receptor at a site different from that of transferrin. When the antibodies were radiolabeled and injected i.v. into cynomolgous monkeys, they distributed selectively to brain but not to other organs or tissues. The antibodies were found almost exclusively in the brain parenchyma, rather than the capillaries, indicating that they had transcytosed the blood-brain barrier. These results show that antibodies to the human transferrin receptor cross the blood-brain barrier and may be useful for noninvasive delivery of therapeutic proteins to the central nervous system.
Subject(s)
Antibodies, Monoclonal/metabolism , Blood-Brain Barrier , Brain/metabolism , Receptors, Transferrin/immunology , Amino Acid Sequence , Animals , Cell Line , Cricetinae , Humans , Macaca fascicularis , Mice , Molecular Sequence Data , Peptides/immunology , Rats , Receptors, Transferrin/metabolism , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
An injectable sustained-release form of human growth hormone (hGH) was developed by stabilizing and encapsulating the protein, without altering its integrity, into biodegradable microspheres using a novel cryogenic process. A single injection of microspheres in monkeys resulted in elevated serum levels of recombinant hGH (rhGH) for more than one month. Insulin-like growth factor-I (IGF-I) and its binding protein IGFBP-3, both of which are induced by hGH, were also elevated for four weeks by the rhGH containing microspheres to a level greater than that induced by the same amount of rhGH administered by daily injections. These results show that, by using appropriate methods of stabilization and encapsulation, the advantages of sustained-release formulations previously demonstrated for low-molecular-weight drugs can now be extended to protein therapeutics.
Subject(s)
Growth Hormone/administration & dosage , Growth Hormone/pharmacokinetics , Animals , Delayed-Action Preparations , Drug Carriers , Growth Hormone/therapeutic use , Humans , Insulin-Like Growth Factor Binding Protein 3/blood , Insulin-Like Growth Factor I/metabolism , Macaca mulatta , Male , Microspheres , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacokinetics , Recombinant Proteins/therapeutic useABSTRACT
We explored the therapeutic potential of a peptide (F20) derived from the filamentous hemagglutinin of Bordetella pertussis in a model of ischemic cell injury after transient (2 hours) middle cerebral artery (MCA) occlusion in the rat. Animals were divided into two groups-(1) F20 peptide group: rats (n = 11) were subjected to 2 hours of transient MCA occlusion, and F20 peptide was administered intravenously (50 nmol) at 0 hours of reperfusion and intraperitoneally (150 nmol/dose) at 2, 4, 6, 8, 22, and 30 hours of reperfusion; (2) control group: rats (n = 10) were administered peptide F23 (a scrambled version of peptide F20) with the same experimental protocol as the F20 peptide group. Forty-six hours after reperfusion, animals were sacrificed, and brain tissue was stained with triphenyltetrazolium chloride for evaluation of tissue damage. To measure neutrophil numbers in ischemic tissue, myeloperoxidase (MPO) immunostaining was performed on a coronal cerebral section in each animal. There was a significant reduction of ischemic infarct volume (36%, p < 0.05) in the F20 group of animals compared with the F23 group. The area of the ischemic lesion was highly correlated with the numbers of the immunoreactive MPO cells (r = 0.78, p < 0.001). The data demonstrate that the F20 peptide significantly reduces infarct volume and intraparenchymal neutrophil numbers after transient MCA occlusion.
Subject(s)
Adhesins, Bacterial/therapeutic use , Bordetella pertussis , Cerebral Infarction/prevention & control , Hemagglutinins/therapeutic use , Ischemic Attack, Transient/drug therapy , Peptides/therapeutic use , Virulence Factors, Bordetella , Adhesins, Bacterial/chemistry , Amino Acid Sequence , Animals , Basal Ganglia/drug effects , Basal Ganglia/enzymology , Basal Ganglia/pathology , Biomarkers , Cerebral Arteries/physiology , Cerebral Cortex/drug effects , Cerebral Cortex/enzymology , Cerebral Cortex/pathology , Cerebral Infarction/pathology , Drug Administration Schedule , Hemagglutinins/chemistry , Ischemic Attack, Transient/pathology , Ischemic Attack, Transient/physiopathology , Least-Squares Analysis , Male , Molecular Sequence Data , Peptides/administration & dosage , Peptides/chemical synthesis , Peroxidase/analysis , Rats , Rats, Wistar , Regression Analysis , Reperfusion , Time FactorsABSTRACT
The integrin CD11b/CD18 promotes leukocyte extravasation during inflammation. Filamentous hemagglutinin (FHA) of Bordetella pertussis binds to CD11b/CD18, raising the possibility that peptides derived from FHA might inhibit leukocyte migration. The Arg-Gly-Asp (RGD) sequence of FHA has been suggested to modulate binding of ligands to CD11b/CD18. Peptides derived from this region inhibited adherence and transendothelial migration of neutrophils in vitro and prevented recruitment of leukocytes into the cerebrospinal fluid in an experimental model of meningitis in rabbits. The mechanism of the antiinflammatory effect may involve modulation of the activity of CD11b/CD18 through peptide interaction with the leukocyte response integrin/integrin-associated protein complex.
Subject(s)
Adhesins, Bacterial/pharmacology , CD18 Antigens/physiology , Cell Adhesion Molecules/biosynthesis , Chemotaxis, Leukocyte/drug effects , Hemagglutinins/pharmacology , Macrophage-1 Antigen/physiology , Neutrophils/physiology , Peptide Fragments/pharmacology , Virulence Factors, Bordetella , Amino Acid Sequence , Animals , Bordetella pertussis/immunology , Cells, Cultured , Dose-Response Relationship, Drug , Endothelium, Vascular , Humans , L-Selectin , Meningitis/blood , Meningitis/cerebrospinal fluid , Meningitis/immunology , Molecular Sequence Data , Neutrophils/drug effects , Oligopeptides , Rabbits , Respiratory Burst , Umbilical VeinsABSTRACT
Factor X (factor ten) of the coagulation cascade binds to the integrin CD11b/CD18 during inflammation, initiating procoagulant activity on the surface of leukocytes (Altieri, D.C., O.R. Etingin, D.S. Fair, T.K. Brunk, J.E. Geltosky, D.P. Hajjar, and T. S. Edgington. 1991. Science [Wash.DC]. 254:1200-1202). Filamentous hemagglutinin (FHA), an adhesin of Bordetella pertussis also binds to the CD11b/CD18 integrin (Relman D., E. Tuomanen, S. Falkow, D.T. Golenbock, K. Saukkonen, and S.D. Wright. 1990. Cell. 61:1375-1382). FHA and the CD11b/CD18 binding loops of Factor X share amino acid sequence similarity. FHA peptides similar to Factor X binding loops inhibited 125I-Factor X binding to human neutrophils and prolonged clotting time. In addition, ETKEVDG and its Factor X analogue prevented transendothelial migration of leukocytes in vitro and reduced leukocytosis and blood brain barrier disruption in vivo. Interference with leukocyte migration by a coagulation-based peptide suggests a novel strategy for antiinflammatory therapy.
Subject(s)
Adhesins, Bacterial , Endothelium, Vascular/drug effects , Factor X/pharmacology , Hemagglutinins/pharmacology , Inflammation/metabolism , Leukocytes/drug effects , Peptide Fragments/pharmacology , Virulence Factors, Bordetella , Amino Acid Sequence , Anti-Inflammatory Agents/pharmacology , Blood Coagulation/drug effects , Blood-Brain Barrier/drug effects , Bordetella pertussis , CD18 Antigens/physiology , Cell Adhesion/drug effects , Cell Adhesion Molecules/pharmacology , Cell Communication/drug effects , Chemotaxis, Leukocyte/drug effects , Factor Xa/drug effects , Humans , Macrophage-1 Antigen/physiology , Molecular Mimicry , Molecular Sequence Data , Monocytes/drug effects , Protein Binding/drug effects , Sequence Homology, Amino AcidABSTRACT
Intrastriatal injections of quinolinic acid induce a pattern of neuronal degeneration similar to that seen in Huntington disease. In the present study, nerve growth factor (NGF) crossed the blood-brain barrier in a dose-dependent fashion following intravenous infusion when conjugated to an antibody directed against the transferrin receptor (OX-26). Intravenous injections of the OX-26-NGF conjugate selectively prevented the loss of striatal choline acetyltransferase-immunoreactive neurons which normally occurs following quinolinic acid administration relative to control rats receiving vehicle or a nonconjugated mixture of OX-26 and NGF. These data demonstrate that a neurotrophic factor-antibody conjugate can prevent the degeneration of central NGF-responsive neurons following systemic administration.
Subject(s)
Corpus Striatum/pathology , Huntington Disease/therapy , Nerve Growth Factors/administration & dosage , Animals , Antibodies, Monoclonal/administration & dosage , Huntington Disease/pathology , Male , Nerve Degeneration , Quinolinic Acid , Rats , Rats, Sprague-Dawley , Receptors, TransferrinABSTRACT
In previous experiments gp160 incorporated into iscom was shown to induce neutralizing antibodies to the homologous as well as the heterologous isolates of HIV-1 (Akerblom et al., AIDS Res., 1991). In the present work we have incorporated into iscoms three defined recombinant DNA products of HIV-1. The carboxy-terminal part of gp120 expressed in E. Coli-PB-1; a chimera containing parts of both p24 and p15 expressed in E. coli-GAG; and baculovirus gp160 cloned in baculovirus and produced in insect cells. Immune responses were induced by the iscom preparations to the homologous antigen as well as to defined recombinant products and to the synthetic peptide RP135 (aa 304-328) harboring a neutralizing epitope. Sera from mice immunized with PB1-iscoms and gp160 (baculo) iscoms were tested in a syncytie inhibition assay. The serum from a mouse immunized with PB1 iscoms reacted strongly with the synthetic peptide RP135 and also neutralized the homologous isolate HIV-1/IIIB with a neutralization titer of 1/64. Three gp160 (baculo) iscom antisera were tested, of which two reacted strongly with the synthetic peptide RP135 but did not neutralize the homologous isolate HIV-1/IIIB. High serum titers were induced in mice by the gp160 iscoms (2 micrograms) to homologous antigen and the recombinant DNA E. coli construct p121 covering part of gp41. The ceilings of the antibody responses were reached after two immunizations. The PB1- and GAG-iscoms required three immunizations to reach the ceiling of the antibody response.
Subject(s)
AIDS Vaccines/immunology , Gene Products, env/immunology , Gene Products, gag/immunology , HIV Antibodies/biosynthesis , ISCOMs/immunology , Nucleocapsid Proteins , Animals , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , HIV Core Protein p24/immunology , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp160 , Mice , Mice, Inbred BALB C , Protein Precursors/immunology , Recombinant Proteins/immunology , Vaccination , gag Gene Products, Human Immunodeficiency VirusABSTRACT
We are beginning to understand the mechanism that envelope proteins of the human and simian immunodeficiency viruses use to gain entry into host cells. A vaccine that can elicit antibodies that bind to the viral epitopes involved in this process would thereby prevent HIV infection. This article outlines our progress in the development of possible candidates for AIDS vaccines.
Subject(s)
AIDS Vaccines/immunology , HIV Antibodies/immunology , HIV Infections/prevention & control , Amino Acid Sequence , Animals , CD4-Positive T-Lymphocytes/immunology , Gene Products, env/immunology , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp160 , HIV-1/immunology , HIV-1/physiology , Humans , Molecular Sequence Data , Neutralization Tests , Protein Precursors/immunology , Simian Immunodeficiency Virus/immunologyABSTRACT
The principal neutralization determinant (PND) of human immunodeficiency virus type 1 envelope glycoprotein gp120 contains a conserved GPG sequence. The effects of a 29-amino-acid deletion of most of the PND, a 3-amino-acid deletion in the GPG sequence, and 16 single-amino-acid substitutions in the GPG sequence were determined in a transient expression assay. All mutant envelope glycoproteins were expressed at levels comparable to that of the wild-type envelope, and mutations in the GPG sequence did not affect processing to gp120 or, except for the 29-amino-acid deletion, binding to CD4. Of all of the mutants, only the GHG and GFG mutants induced formation of syncytia similar in size and number to those induced by the wild-type envelope. When the envelope expression level was increased 10-fold or more, several additional mutants (APG, GAG, GSG, GQG, GVG, and GPF) also induced syncytium formation. Transfection with infectious proviral molecular clones containing the GHG, GFG, APG, GAG, GSG, or GPF mutations induced production of viral particles; however, only the GPG, GHG, and GFG viruses produced active infections in CD4-bearing cells. Furthermore, whereas the wild-type virus was efficiently neutralized by PND polyclonal and monoclonal antibodies, the GHG- and GFG-containing viruses were not. These results show that mutations in the GPG sequence found within the PND do not affect envelope expression and do not significantly affect CD4 binding or production of viral particles but that they do affect the ability of the envelope to induce syncytia and those of the viral particles to infect CD4 cells and be neutralized by PND antibodies.
Subject(s)
Giant Cells , HIV Envelope Protein gp120/genetics , HIV-1/genetics , Mutation , Peptide Fragments/genetics , Amino Acid Sequence , Base Sequence , Blotting, Western , CD4 Antigens/metabolism , Cell Line , DNA, Viral , HIV Antibodies/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/immunology , HIV-1/growth & development , HIV-1/immunology , HIV-1/physiology , Humans , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Neutralization Tests , Peptide Fragments/chemistry , Peptide Fragments/immunology , Proviruses/immunology , Radioimmunoprecipitation Assay , Transfection , Virus Replication/geneticsABSTRACT
The entry of human immunodeficiency virus type 1 into two T-cell lines has been analyzed to determine the relative time courses with which virus entry can be blocked (i) by washing, (ii) by adding a monoclonal antibody to the V3 loop of gp120 that neutralizes without blocking CD4 binding (0.5 beta), or (iii) by adding an antireceptor monoclonal antibody that competes for virus binding (leu3a). During entry into C8166 cells, 50% escape from the wash as well as the anti-V3 loop antibody required 20 min, whereas 50% escape from the leu3a block required 45 minutes. In contrast, during entry into H9 cells, 50% escape from the wash block required 50 min, 50% escape from the anti-V3 loop antibody required 110 min, and 50% escape from the antireceptor antibody required 190 min. These results demonstrate that the times required for entering virus to escape each of the blocks were cell type specific. They also demonstrate that V3 loop-dependent steps occur relatively early in entry and suggest that binding of gp120 to CD4 is important for late as well as early steps in human immunodeficiency virus type 1 entry.