Immunochemical mapping of .alpha.-2 interferon

Abstract

A panel of five monoclonal antibodies, designated U1-U5, produced by murine hybridoma clones has been raised to recombinant interferon (IFN) alpha-2, and one monoclonal antibody, designated U6, has been raised to a mixture of cyanogen bromide fragments of IFN alpha-2. These antibodies have been characterized with respect to (1) neutralization of IFN antiviral and antiproliferative activities, (2) binding to four cloned IFN alpha subtypes (alpha-1, alpha-2, alpha-4, and alpha-7) that are naturally occurring and to two novel products of recombinant DNA technology (delta-4 alpha-1 and delta-4 alpha-2/alpha-1 hybrid), and (3) binding to three cyanogen bromide fragments of IFN alpha-2. Four of the six monoclonal antibodies inhibited IFN antiviral activity. In conjunction with the previously reported monoclonal antibodies III/21 [Arnheiter, H., Thomas, R. M., Leist, T., Fountoulakis, M., & Gutte, B. (1981) Nature (London) 294, 278-280] and NK-2 [Secher, D. S., & Burke, D. C. (1980) Nature (London) 285, 446-450], eight unique epitopes have been described. Analysis of cross-reactivity patterns with IFN alpha fragments and subtypes indicated that monoclonal antibodies U1 and NK-2, which neutralized both antiviral and antiproliferative activities, and U2, which was nonneutralizing in these assays, were directed to distinct epitopes located in a polypeptide consisting of the amino-terminal 15 amino acid residues linked to residues 60-110 by a disulfide bond. The epitope recognized by U1 was determined to reside, at least in part, between residues 5 and 15. Competitive binding studies indicated that neutralizing monoclonal antibody U3, which did not bind to any of the cyanogen bromide fragments, was directed to an epitope partially overlapping that of NK-2. Epitopes to which neutralizing monoclonal antibodies U3, U4, and U5 and nonneutralizing antibody U6 were directed were readily distinguished by cross-reactivity with IFN alpha subtypes. The nonneutralizing monoclonal antibody U6 was determined to be directed to an epitope between residues 22 and 58. The fact that delta-4 alpha-1 and the delta-4 alpha-2/alpha-1 hybrid were active in an antiviral assay indicated a lack of direct functional significance for the first four amino-terminal amino acid residues and the Cys1-Cys98 disulfide bond. However, reduction with 2-mercaptoethanol of IFN alpha-2 altered the integrity of four of the eight epitopes. These data support a critical role for disulfide linkages in maintaining the native conformation of IFN alpha-2 and provide a potential basis for predicting the location of functionally important domains.

Dendritic cells from HIV-1 infected individuals are less responsive to toll-like receptor (TLR) ligands

“…Custom conjugates of mouse anti-human monoclonal antibodies against IFN-α PE (clone 7N4-1) and CD123 PerCP-Cy5.5 were also provided by BD Biosciences. The 7N4-1 antibody reacts with human IFN-α 2b (31,32) and to a lesser extent IFN-α 7 (33). It does not react with IFN-α 1 or IFN-α 4 (33).…”

“…The 7N4-1 antibody reacts with human IFN-α 2b (31,32) and to a lesser extent IFN-α 7 (33). It does not react with IFN-α 1 or IFN-α 4 (33). Matched fluorchrome-conjugated isotype-control antibodies were used in these studies.…”

Evolution of MDA-5/RIG-I-dependent innate immunity: Independent evolution by domain grafting

“…The relationship between IFN and viral infection is well documented and supported by studies employing neutralizing antibodies and IFN-receptor knockout mice, which document a direct role for IFN in mediating host defense against viruses (7,8). Moreover, IFNs are now recognized as multifunctional molecules that also provide defense against bacterial infection (especially intracellular parasites), induce antitumor activity (both direct and immune system-mediated), and stimulate or inhibit differentiation depending on cellular context (5, 9).…”

Antibodies that neutralize human beta interferon biologic activity recognize a linear epitope: analysis by synthetic peptide mapping.

“…Previous structure-function studies have utilized genetically altered IFN gene products (2)(3)(4)(5)(6)(7)(8), peptide mapping of large fragments (9)(10)(11)(12)(13)(14), and immunochemical mapping with monoclonal antibodies (mAbs) (15)(16)(17)(18). Such analyses of human a and p interferon (hIFN-a and -4) molecules have yielded inconclusive results, implicating large regions near the amino terminus (2,3,7,9,15), carboxyl terminus (11,16), or both (4-6, 8, 10), as domains responsible for biologic activity. The screening of short, sequential overlapping peptides for antibody reactivity as described by Geysen et al (19,20), also known as "pepscan" (21), permits the simultaneous scanning of entire molecules for linear immunoreactive epitopes.…”