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D Healey

Publications and source records attributed to D Healey.

33 records · Page 2Linked to original sources
PubMed1992

Construction of a binding site for human immunodeficiency virus type 1 gp120 in rat CD4.

The human immunodeficiency virus (HIV-1) infects T lymphocytes via an interaction between the virus envelope glycoprotein gp120 and the CD4 antigen of T helper cells. Previous studies demonstrated that mutations in various regions of CD4 domain 1 lead to the loss of gp120 binding. In the present study the gp120 binding site was constructed in rat CD4 by replacing rat with human CD4 sequence. A series of mutants was constructed the best of which bound gp120 with an affinity only twofold less than that of human CD4. The data indicate that the gp120 binding site of human CD4 is constituted by residues 33-58 of domain 1.

Amino Acid Sequence↗
PubMed1991

A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion.

The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C'' and FG. This structure is consistent with C'C'' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.

Antibodies, Monoclonal↗
PubMed1990

Novel anti-CD4 monoclonal antibodies separate human immunodeficiency virus infection and fusion of CD4+ cells from virus binding.

Human immunodeficiency virus (HIV) binds to cells via an interaction between CD4 and the virus envelope glycoprotein, gp120. Previous studies have localized the high affinity binding site for gp120 to the first domain of CD4, and monoclonal antibodies (mAbs) reactive with this region compete with gp120 binding and thereby block virus infectivity and syncytium formation. Despite a detailed understanding of the binding of gp120 to CD4, little is known of subsequent events leading to membrane fusion and virus entry. We describe two new mAbs reactive with the third domain of CD4 that inhibit steps subsequent to virus binding critical for HIV infectivity and cell fusion. Binding of recombinant gp120 or virus to CD4 is not inhibited by these antibodies, whereas infection and syncytium formation by a number of HIV isolates are blocked. These findings demonstrate that in addition to virus binding, CD4 may have an active role in membrane fusion.

Animals↗
PubMed1989

Structural analysis of the human immunodeficiency virus-binding domain of CD4. Epitope mapping with site-directed mutants and anti-idiotypes.

The CD4 molecule, a differentiation marker expressed primarily by T lymphocytes, plays an important role in lymphocyte activation. CD4 is also the receptor for HIV. A number of recent studies have localized the high affinity binding site of the HIV envelope glycoprotein, gp120, to the NH2-terminal (V1) domain of CD4, a region with sequence and predicted structural homology with Ig kappa chain V domains (V kappa). In this report, we show that V1 bears structural similarities with V kappa regions through detailed epitope mapping of 26 CD4 mAbs. The binding sites of these mAbs were initially defined relative to one another by crossblocking analysis and were then localized to specific domains of CD4 in blocking studies with truncated, soluble CD4 proteins. The epitopes within the V1 domain were mapped in detail with a panel of 17 substitution mutants, and the specificities of several mAbs that appear to recognize very similar epitopes were examined in crossblocking studies with anti-idiotype antibodies. The location of the epitopes is consistent with a V kappa-like structure of V1. Most of the epitopes lie within regions of predicted exposed loops. A number of these epitopes span discontinuous residues in the linear sequence that lies in close proximity in an Ig fold. Alignment of CD4 V1 with the Ig V kappa chains places these epitopes within stretches corresponding to the complimentarity-determining regions. This epitope analysis is relevant for a vaccine strategy for HIV based on anti-idiotype antibodies to CD4 mAbs and for studies with CD4 antibodies on the role of CD4 in T lymphocyte activation.

Amino Acid Sequence↗
PubMed1989

Limitations of the anti-idiotype strategy for an HIV vaccine.

Attempts to raise protective immunity to HIV have been notably ineffective. However the conservation of binding of different virus strains to CD4 suggests that the HIV envelope glycoprotein (gp120) should have a conserved site for CD4. Attempts to raise neutralizing anti-idiotypes to CD4 monoclonal antibodies (MoAbs) have generated polyclonal sera which block HIV-induced syncytium formation in vitro but have low titres. Mapping of CD4 epitopes recognized by CD4 MoAbs and gp120 indicates that none of the present CD4 MoAbs bind to exactly the same site as gp120, which may explain the relative lack of success of the anti-idiotype approach to date.

Animals↗
PubMed1986

Evaluation of a new assay for antibodies to LAV/HTLV III.

The sensitivity, specificity and reproducibility of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to LAV/HTLV III produced by Genetic Systems was assessed with the identical panel of sera used in previous evaluations of anti-HTLV III ELISAs. The results from this study show that the Genetic Systems anti-LAV/HTLV III ELISA proved to be of equivalent sensitivity and to have higher specificity than assays currently used in Australia for screening purposes while maintaining high levels of intra- and inter-laboratory reproducibility.

Antibodies, Viral↗
PubMedDate not supplied

A multi-institutional phase II study of BMS-182248-01 (BR96-doxorubicin conjugate) administered every 21 days in patients with advanced gastric adenocarcinoma.

PURPOSE: High levels (> 200,000 molecules per carcinoma cell) of the LewisY antigen are expressed on the surface of most (> 75%) gastric carcinomas. The BMS-182248-01 is a chimeric variant of anti-LewisY monoclonal antibody that is conjugated with doxorubicin. In a phase I study, BMS-182248-01 resulted in a partial response in a patient with gastric carcinoma. We, therefore, conducted a multi-institutional phase II study of BMS-182248-01 in patients with advanced gastric carcinoma. METHODS AND PATIENTS: Only patients with evidence of LewisY antigen by immunohistochemical method on their gastric carcinoma were treated. Patients with unresectable gastric adenocarcinoma were eligible. Patients had to have adequate liver, renal, and marrow functions. Written consent was obtained from all patients. All patients were hospitalized. BMS-182248-01 was administered at the starting dose of 700 mg/m2 i.v. over 24 hours on day 1 every 3 weeks. RESULTS: Fifteen patients were enrolled. There were 10 men and 5 women. The median age at enrollment was 56 years, with ages ranging from 34 to 80 years. No objective responses were observed. Five patients had disease stabilization. The remaining 10 patients progressed on study. Rapidly reversible gastrointestinal toxicity, primarily nausea and emesis, was predominant. There was no neutropenia, thrombocytopenia, or cardiomyopathy. CONCLUSIONS: Although BMS-182248-01 represents a novel approach of monoclonal antibody conjugated with an active chemotherapy agent, delivered intracellularly, it was ineffective in patients with gastric carcinoma whose tumors carried LewisY antigen.

Adenocarcinoma↗