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The vast majority of human rhinovirus serotypes utilize the intercellular adhesion molecule 1 (ICAM-1) as the attachment site on susceptible cells. Twelve murine monoclonal antibodies were isolated and shown by competition binding studies to recognize three distinct, nonoverlapping epitopes on the ICAM-1 receptor. Titration of three antibodies representing each of the binding sites demonstrated that they were equally effective at blocking viral attachment. By using in vitro transcription and translation systems, a series of progressive C-terminal truncations of ICAM-1 molecules was generated. Immunoprecipitation of these fragments with each of the three antibodies indicated that all three epitopes reside within the first 82 amino acids of the receptor. Attempts to demonstrate specific binding of these in vitro-synthesized receptor fragments to virions were unsuccessful. The inability to show virion binding was most likely due to a failure of the lysates to properly glycosylate the receptor molecule, since native, unglycosylated receptor molecules isolated from cell membranes were also inactive in virus binding assays.

BALB/c mice were immunized with purified preparations of hepatitis A virus (HAV) isolated after 21 days of growth in LLC-MK2 cells. The HAV antigen was isolated from CsCl gradients and consisted primarily of the following three proteins as analyzed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining: VP-1 at 33,000 daltons, VP-2 at 29,000 to 30,000 daltons, and VP-3 at 27,000 daltons. The spleen cells isolated from two BALB/c mice, immunized with two inoculations of HAV, were fused with SP 2/0 myeloma cells and grown in hypoxanthine-aminopterin-thymidine medium. Of 270 hybridomas initially screened, 72 were positive for binding HAV by a noncompetitive radioimmunoassay. All 72 were tested for the ability to neutralize the infectivity of HAV in an in vitro cell culture assay that was adapted for microtiter plates and that used detergent-treated virus for improved neutralization sensitivity and newborn cynomolgus monkey kidney cells for rapid growth. Eighteen hybridomas were positive for neutralization; 16 remained stable. Of the 16, 9 were able to compete with labeled polyclonal serum for binding to HAV. The nine competing hybridomas could be separated into two groups which appear to be directed towards two different sites on HAV and could complement each other in the competitive radioimmunoassay against polyclonal sera. Of the original 16 neutralizing hybridomas, 4 were subcloned through two cycles of limit dilutions. All four monoclonal antibodies retained their original neutralizing and competitive properties; three were immunoglobulin G2a, and one was immunoglobulin G1. All four monoclonal antibodies readily precipitate whole 125I-labeled HAV but are not able to recognize the disrupted proteins of the virus (as tested by immune precipitations of heat- and detergent-disrupted virions or Western blot analyses). However, the heterobifunctional cross-linking reagent toluene-2,4-diisocyanate was used to cross-link purified Fab fragments of two different monoclonal antibodies (2D2 and 6A5) to HAV before disruption. This reagent demonstrated a specific reaction of the monoclonal antibodies to the VP-1 of HAV, suggesting this major surface protein contains at least one of the major neutralization sites for HAV.

Human rhinoviruses can be classified into major and minor groups on the basis of receptor specificity. Recently, a mouse monoclonal antibody was isolated which selectively blocked the attachment of the major group of human rhinoviruses to cells. Using this monoclonal antibody, the cellular receptor for the major group of human rhinoviruses was isolated. A radioimmunoassay was developed by using the receptor antibody to specifically detect rhinovirus receptor during isolation. Solubilized receptor from detergent-treated HeLa cell membrane extracts eluted from gel filtration columns with an apparent molecular weight of 440,000. A cellular receptor protein, which had a molecular weight of 90,000 when analyzed on sodium dodecyl sulfate-polyacrylamide gels, was purified from solubilized extracts on an immunoaffinity column containing receptor antibody. Polyclonal rabbit antiserum, resulting from immunization with the isolated receptor protein, specifically blocked the attachment of the major group of human rhinoviruses and indicated that the 90-kilodalton protein plays a functional role in attachment. Prolonged exposure of HeLa cell monolayers with the receptor antibody showed no inhibition of cell growth and division.

Previous molecular and immunological studies have mapped four neutralization sites on human rhinovirus type 14 (B. Sherry, A. G. Mosser, R. J. Colonno, and R. R. Rueckert, J. Virol. 57:246-257, 1986). Eight monoclonal antibodies, one pair for each of the four target sites and all belonging to a single isotype, immunoglobulin G2a, were studied under conditions which resulted in 95% neutralization of infectious viral particles. All eight antibodies shifted the isoelectric point of virions from 6.7 to much more acidic forms, ranging from pI 1.8 to 3.2. In addition, antibodies targeted against three of the four neutralization sites caused significant aggregation of virions under the neutralization conditions employed. Aggregation could be reversed by digesting virus-antibody complexes with papain. Following papain digestion, the acidic pIs of three of the neutralized virus preparations returned to neutral and infectivity was restored. Membrane-binding assays with virus neutralized with a nonaggregating antibody showed a dose-related inhibition of virus attachment to cellular receptors. Purified Fab fragments at a 13- to 61-fold-higher concentration than intact antibodies caused a comparable isoelectric shift, neutralized virions in the absence of aggregation, and interfered with attachment of virions to host cell receptors in a membrane-binding assay. These findings suggest that neutralizing antibodies interfere with the attachment of rhinoviruses to cellular receptors and that bivalent attachment of antibody is not a prerequisite for neutralization.