Vincent J. Merluzzi

A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses

Rhinoviruses, which cause common colds, possess over 100 serotypes, 90% of which (the major group) share a single receptor. Lymphocyte function associated molecule 1 (LFA-1) mediates leukocyte adhesion to a wide variety of cell types by binding to intercellular adhesion molecule 1 (ICAM-1). We demonstrate identity between the receptor for the major group of rhinoviruses and ICAM-1. A major group rhinovirus binds specifically to purified ICAM-1 and to ICAM-1 expressed on transfected COS cells, and binding is blocked by three ICAM-1 monoclonal antibodies (MAb) that block ICAM-1-LFA-1 interaction, but not by an ICAM-1 MAb that does not block ICAM-1-LFA-1 interaction. This suggests that the ICAM-1 contact site(s) for LFA-1 and rhinoviruses is proximal or identical. In addition, ICAM-1 MAb block the cytopathic effect in HeLa cells mediated by representative major but not minor group rhinoviruses. ICAM-1 is induced by soluble mediators of inflammation, suggesting that the host immune response to rhinovirus may facilitate spread to uninfected cells.

Reduction in the severity of graft-versus-host disease and increased survival in allogenic mice by treatment with monoclonal antibodies to cell adhesion antigens LFA-1 alpha and MALA-2.

Bone marrow transplantation is a therapeutic treatment for many life-threatening hematologic disorders, especially leukemia and certain immune deficiency diseases. However, acute graft-versus-host disease is often associated with bone marrow transplantation. In mice, allogeneic GVHD appears to be mediated by both host natural killer cells and donor T cells. In vitro and in vivo experiments demonstrate that treatment with either YN1/1.7 or M17/4.2 mabs is immunomodulatory and inhibits both the mixed lymphocyte reaction and natural killer cell activity. In addition, utilizing an allogeneic model of acute, lethal GVHD with C57B1/6 mice as donors and sublethally irradiated BDFi mice as recipients, treatment of host mice with anti-LFA-lα (Ml7/ 4.2) or anti-MALA-2 (YN1/1.7) mabs at a dose of 10 mg/kg/day for 10 days significantly reduced GVHD and enhanced survival. Mabs to lymphocyte adhesion molecules such as LFA-lα and MALA-2 may provide a useful therapy for the treatment of GVHD.

Polypeptide 2A of human rhinovirus type 2: identification as a protease and characterization by mutational analysis

Evidence is presented that the protein 2A of human rhinovirus serotype 2 (HRV2) is a protease. On expression of the VP1-2A region of HRV2 in bacteria, protein 2A was capable of acting on its own N-terminus; derived extracts specifically cleaved a 16 amino acid oligopeptide corresponding to the sequence at the cleavage site. Cleavage of the oligopeptide substrate provides a convenient in vitro assay system. Deletion experiments showed that removal of 10 amino acids from the carboxy terminus inactivated the enzyme. Site-directed mutagenesis identified an essential arginine close to the C-terminus and showed that the enzyme was sensitive to changes in the putative active site. This analysis supports the hypothesis that 2A belongs to the group of sulfhydryl proteases, although sequence comparisons indicate that the putative active site of HRV2 2A is closely related to that of the serine proteases.

Monoclonal antibody to MALA-2 (ICAM-1) reduces acute autoimmune nephritis in kdkd mice

Hereditary tubulointerstitial nephritis is a prominent cause of renal failure in humans. A variety of animal models utilizing immunologically induced nephritis have been developed. The kdkd congenic variant of the CBA/Ca mouse has normal kidneys at birth but develops progressive, lethal autoimmune nephritis beginning at approximately Week 8. The destruction of renal tubular epithelium in mediated by a population of antigen-specific, H-2Kk-restricted, Lyt-2+, L3T4- T cells. The present experiments demonstrate that systemic treatment with anti-ICAM-1 monoclonal antibody reduces kidney disease in kdkd mice. Anti-ICAM-1 mab localizes to inflammatory sites in the kidney and effects a significant reduction in leukocyte infiltration. Concomitantly, urine protein levels of anti-ICAM-1-treated mice are significantly reduced. The use of anti-adhesion molecule monoclonal antibodies that alter leukocyte activity and/or trafficking may be useful therapies for certain autoimmune disorders.

Monoclonal antibodies to lymphocyte function-associated antigen-1 inhibit invasion of human lymphoma and metastasis of murine lymphoma

The leukocyte integrins are cell adhesion molecules which play pivotal roles in the development of a variety of immune responses including T-cell-mediated cytotoxicity, lymphocyte proliferation, macrophage presentation of antigen, and adhesion of leukocytes to vascular endothelium. The relevance of lymphocyte function-associated antigen-1 (LFA-1) to leukocyte malignancies is currently under examination in a number of laboratories. Here, we present evidence demonstrating that LFA-1 plays a role during the in vitro invasion of human endothelium by JY lymphoma cells and during in vivo metastasis of two distinct models of murine leukemia: P815 mastocytoma and EL4 lymphoma. When assayed in vitro, a murine anti-human LFA-1 (α subunit) monoclonal antibody (mAb) inhibits up to 80% of JY lymphoma cell invasion. When assayed in vivo, a rat anti-LFA-1 (a subunit) mAb significantly inhibited the development of experimental metastases, when administered concomitantly with either P815 or EL4 tumor cells. The leukocyte integrins, particularly LFA-1, may represent useful targets for the therapeutic modulation of metastasis.

Distribution of ICAM-1 Within Decidua and Placenta and its Gestational Age-Associated Changes

Intercellular adhesion molecule 1 (ICAM-1), a ligand of leukocyte function associated antigen 1 (LFA-1), is present on many cells, including monocyte/macrophages. ICAM-1 is considered to play an important role in the induction and maintenance of inflammatory responses by permitting leukocyte adhesion. Its expression is inducible on endothelial and epithelial cells exposed to various inflammatory cytokines (preceding expression of HLA-DR) and is maturation dependent in certain cell lines. The distribution of ICAM-1 in decidua and placenta was evaluated using peroxidase-antiperoxidase immunohistochemistry. In decidua of first and third trimesters, scattered ICAM-1-staining cells were observed. In placentas of first and third trimesters, all types of trophoblasts stained negatively for ICAM-1. Prior to 10 weeks of gestation, the villous stroma was uniformly ICAM-1 and HLA-DR unreactive. Beginning in the chorionic plate at approximately 10 weeks, scattered ICAM-1-positive stromal cells were observed, whereas stromal cells of the terminal villi revealed no ICAM-1. By 14-16 weeks, approximately 40-50% of the terminal villous stromal cells were ICAM-1 staining. This parallels the 40-50% of the villous stromal cells that share other immunohistochemical markers, such as EB-11, with monocyte/macrophages. The lack of functional maturation of the villous stromal macrophage may explain the rarity of chronic villitis early in gestation.

Discovery of Nevirapine, a Nonnucleoside Inhibitor of HIV-1 Reverse Transcriptase

Five years after the description of acquired immunodeficiency syndrome (AIDS) and approximately 3 years after the discovery of human immunodeficiency virus type 1 (HIV-1), we began a study group around the therapeutic target of HIV-1 protease (a virus-specific enzyme involved in processing and maturation). The goals of this group were to study the HIV-1 protease, its substrates, and use of possible peptide inhibitors. During this time a few of us discussed the possibility of targeting HIV-1 reverse transcriptase (RT) as a target for therapeutic intervention. It was clear that resources could not be spread too thin and it was also clear to us that we did not want to design or rediscover nucleoside analogues as inhibitors. In fact, if we were to embark on an inhibitor program for RT, our foremost objective was to search for and design inhibitors that were clearly nonnucleoside in nature. RT seemed to be an excellent target because it was known to be unique to retroviruses (an exception being hepatitis B) and not usually present in normal cells. Yet, the only true worthwhile inhibitors of this enzyme were nucleoside analogues of which AZT (zidovudine) was the prototype and soon after the only approved drug for HIV-1. It was clear to some of us that we should somehow target HIV-1 RT but in a way that resources, equipment, and personnel use did not detract from ongoing projects and in particular the HIV-1 protease research team. Since this was a chance venture, it was decided that we would design an RT “screen” using an established assay system and randomly test all compounds available in the worldwide Boehringer Ingelheim compound repositories. If any true positive compounds were identified as inhibitors, we would then rationally search for analogues and start structure-activity relationship (SAR) based synthesis here in the United States. It was necessary from the beginning to identify which RT to use and how to reassure ourselves of finding a true inhibitor, that is, specific for RT only. We decided from the very beginning not to address virus replication but rather to concentrate on inhibitors of the RT enzyme and address the question of viral replication later. This meant, of course, that an inhibitor, once discovered, might or might not work on the virus. Problems that could arise might be an inability to penetrate the cell, solubility in culture medium, cell metabolism of the compound, and generally unforseen metabolic problems. Yet, we felt that if a specific RT inhibitor could be found, cell penetration and other problems could be overcome by synthetic design.

Detection of major group rhinoviruses by soluble intercellular adhesion molecule-1 (sICAM-1).

Soluble intercellular adhesion molecule-1 (sICAM-1) was shown to be the receptor for the major subgroup of rhinoviruses. It was demonstrated that this molecule can inhibit the binding and subsequent infection of target cells by rhinoviruses belonging to the major but not the minor subgroup. The data reported now describe an ELISA-based system utilizing biotinylated sICAM-1 as a means of detecting rhinoviruses belonging to the major subgroup.

A Novel, Non-Nucleoside Inhibitor of HIV-1 Reverse Transcriptase (Review)

Human immunodeficiency virus type 1 (HIV-1) is the retrovirus responsible for the majority (>95%) of the acquired immunodeficiency syndrome (AIDS) cases in the world. The complicated life-cycle of this virus presents many challenging areas for intervention. Our laboratories have concentrated on prevention of the early phase in proviral synthesis, specifically interruption of the RNA > DNA metabolic process by interfering with the viral enzyme, reverse transcriptase (RT). The RT of HIV-1 is a necessary component for early proviral synthesis. This enzyme has binding sites for nucleoside triphosphates, template primer and a catalytic site for the polymerase reaction. Nucleotides are added to the polymerizing chain to create a complementary DNA molecule (for review see Gilboa et al., 1979). The most effective inhibitors of RT have been the nucleoside analogs which are converted to triphosphates by cellular enzymes and act as chain terminators of the RT reaction (Mitsuya et al., 1985). Zidovudine (3’-azido-2’-3’dideoxy-thymidine, AZT) has been shown to be of benefit in HIV-1 infected individuals (Yarchoan et al., 1986). However, there are side-effects associated with the use of AZT (Richman et al., 1987), in addition to incomplete viral inhibition (Ho et al., 1989) and viral resistance (Larder et al. 1989).