ABSTRACT
A strategy for HIV-1 vaccine development is to define envelope (Env) evolution of broadly neutralizing antibodies (bnAbs) in infection and to recreate those events by vaccination. Here, we report host tolerance mechanisms that limit the development of CD4-binding site (CD4bs), HCDR3-binder bnAbs via sequential HIV-1 Env vaccination. Vaccine-induced macaque CD4bs antibodies neutralize 7% of HIV-1 strains, recognize open Env trimers, and accumulate relatively modest somatic mutations. In naive CD4bs, unmutated common ancestor knock-in mice Env+B cell clones develop anergy and partial deletion at the transitional to mature B cell stage, but become Env- upon receptor editing. In comparison with repetitive Env immunizations, sequential Env administration rescue anergic Env+ (non-edited) precursor B cells. Thus, stepwise immunization initiates CD4bs-bnAb responses, but immune tolerance mechanisms restrict their development, suggesting that sequential immunogen-based vaccine regimens will likely need to incorporate strategies to expand bnAb precursor pools.
Subject(s)
Antibodies, Neutralizing/biosynthesis , B-Lymphocytes/immunology , HIV Antibodies/biosynthesis , HIV-1/immunology , env Gene Products, Human Immunodeficiency Virus/immunology , AIDS Vaccines/immunology , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , B-Lymphocytes/cytology , Binding Sites, Antibody , CD4 Antigens/metabolism , Cell Lineage/immunology , Clonal Anergy , Female , Gene Knock-In Techniques , HIV Antibodies/chemistry , HIV Antibodies/genetics , Humans , Immune Tolerance , Immunization/methods , Macaca mulatta , Male , Mice , Mice, Transgenic , Models, MolecularABSTRACT
Exposure to atherogenic levels of low-density lipoprotein (LDL) causes elevated reactive oxygen species (ROS) production by human endothelial cells (ECs). NADPH oxidase is thought to be the main source of ROS generated by LDL-activated ECs. The mechanism by which this lipoprotein activates endothelial NADPH oxidase is incompletely understood. To gain further insight into the signaling pathway, the authors have examined the effects of inhibitors to various signal transducing enzymes, including the G(i)-protein coupled receptor (pertussis toxin), Src tyrosine kinase (PP1), phospholipase C-gamma (U73122), phosphatidylinositol 3-kinase (LY294002), p42/p44 mitogen-activated protein kinase (MAPK) kinase (PD98059), p38 MAPK (SB203580), protein kinase C (Ro 318220, GF 109203X, Go 6976), and cytosolic phospholipase A(2) (AACOCF3), on the ROS-producing capacity ECs activated by LDL. Exposure of cultured ECs to LDL (0.45 mg protein/mL) stimulated ROS formation, as measured using a 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate assay. This effect was partially inhibited by Ro 318220, GF 109203X, U73122, and SB203580, and blocked or nearly completely inhibited by PP1, pertussis toxin, LY294002, PD98059, and AACOCF3. Only a partial, minor inhibition occurred with the protein kinase C inhibitor, Go 6976. These results are most consistent with LDL activating endothelial NADPH oxidase, predominantly through a signaling pathway that leads to cytosolic phospholipase A(2) activation.
Subject(s)
Endothelium, Vascular/enzymology , Lipoproteins, LDL/metabolism , NADPH Oxidases/metabolism , Arachidonic Acids/pharmacology , Cells, Cultured , Chromones/pharmacology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Estrenes/pharmacology , Flavonoids/pharmacology , Humans , Imidazoles/pharmacology , Indoles/pharmacology , Maleimides/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Morpholines/pharmacology , NADPH Oxidases/drug effects , Pertussis Toxin , Phosphatidylinositol 3-Kinases/metabolism , Phospholipase C gamma , Phospholipases A/metabolism , Protein Kinase C/metabolism , Pyridines/pharmacology , Pyrrolidinones/pharmacology , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Type C Phospholipases/metabolism , Umbilical Veins/cytology , p38 Mitogen-Activated Protein Kinases , src-Family Kinases/metabolismABSTRACT
NADPH oxidase is a major enzymatic source of oxygen free radicals in stimulated endothelial cells (ECs). The ortho-methoxy-substituted catechol, apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa, inhibits the release of superoxide anion (O2*-) by this enzyme. The compound acts by blocking the assembly of a functional NADPH oxidase complex. The underlying chemistry of this inhibitory activity, and its physiological significance to EC proliferation, have been investigated. A critical event is the reaction of ortho-methoxy-substituted catechols with reactive oxygen species (ROS) and peroxidase. Analysis of this reaction reveals that apocynin is converted to a symmetrical dimer through the formation of a 5,5' carbon-carbon bond. Both reduced glutathione and L-cysteine inhibit this dimerization process. Catechols without the ortho-methoxy-substituted group do not undergo this chemical reaction. Superoxide production by an endothelial cell-free system incubated with apocynin was nearly completely inhibited after a lagtime for inhibition of ca. 2 min. Conversely, O2*- production was nearly completely inhibited, without a lagtime, by incubation with the dimeric form of apocynin. The apocynin dimer undergoes a two-electron transfer reaction with standard redox potentials of -0.75 and -1.34 V as determined by cyclic voltammetry. Inhibition of endothelial NADPH oxidase by apocynin caused a dose-dependent inhibition of cell proliferation. These findings identify a metabolite of an ortho-methoxy-substituted catechol, which may be the active compound formed within stimulated ECs that prevents NADPH oxidase complex assembly and activation.
