Inhibition of cell adhesion to the virus by synthetic peptides of fiber knob of human adenovirus serotypes 2 and 3 and virus neutralisation by anti-peptide antibodies

Abstract

The fiber knob of adenovirus (Ad) causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s) several synthetic peptides derived from Ad2 and Ad3 fiber head sequences and their antisera were tested for interference with virus attachment to HeLa and FL cells and cell adhesion to viruses. The anti-peptide sera were also evaluated in ELISA and virus neutralisation test. Ad2 (of subgroup C) and Ad3 (of subgroup B) attachment was not significantly inhibited by peptides corresponding to the amino acid residues 535-554, 555-573, 562-582 of Ad2 fiber or 210-225, 267-283, 291-306 and 300-319 of Ad3 fiber. However, microplate pre-adsorbed Ad3 fiber residues 210-225 and 267-283 could bind FL and HeLa cells, and 1 mg/ml of Ad3 fiber residues 267-283 inhibited the cell adhesion to Ad3 virus to approximately 90%. This peptide may participate in the receptor binding site of Ad3 fiber. ELISA reactive anti-peptide antibodies against the homologous peptide and virus did not significantly reduce the cell adhesion to the immobilised virus or the virus attachment to cells, but in the neutralisation assay antibodies raised to Ad2 fiber residues 555-573 and 562-582 and Ad3 fiber residues 210-225 caused neutralisation of the homologous virus at serum dilutions of 1:500 and 1:32, respectively. The corresponding peptides and one further peptide of Ad2 fiber and two of Ad3 fiber seem to contain neutralisation epitopes.

Neutralizing Antibody Blocks Adenovirus Infection by Arresting Microtubule-Dependent Cytoplasmic Transport

“…Using several approaches, it has been shown convincingly that hexon is the major target for neutralization (8,14,28,34,45). Antifiber and anti-penton base NAbs that block primary receptor interactions and integrin-mediated internalization, respectively, have been identified (7,13,19,32,45); however, they likely represent only a small portion of the neutralizing activity of serum. Studies of the mechanisms of antiHAdV NAbs have identified modes of action that are common to antibody neutralization of other viruses.…”

Mapping of Epitopes on the Fiber Knobs of Human Adenovirus Serotypes 8 and 15

“…We used native conditions as recommended by the manufacturer for the spin columns (Clontech, Heidelberg, Germany). The antisera were produced as previously described [8,19].…”

“…For antisera production, serotypes Ad3, Ad4, Ad5 and Ad12 (from Dr. T. Adrian), Ad8 (from Prof. G. Wadell, Department of Virology, Umeå University, Sweden), Ad15 and Ad41 (from American Type Culture Collection, Rockville, Md., USA) were propagated in FL or HeLa cells, purified by CsCl density gradient centrifugation and dialyzed against phosphatebuffered saline, pH 7.4, containing 10% glycerol, and the virus content was determined by UV absorption measurement [8,19,23].…”

“…Also, some subgenus BI and BII fiber knob-derived peptide-specific antisera were able to inhibit the homologous adenovirus infection of cells [14,19]. Therefore, we evaluated the two first longer peptides FK15 (aa 231-255), spanning the reactive peptides P10 (partly) and P12-P14, and FK15 (aa 308-332), spanning the reactive peptides P26 (partly) and P29, for immunogenicity, because these sequences represent epitopes.…”

“…Such 25-mer peptides are potentially able to induce antibodies without carrier conjugation. Antisera specific to our 25-mer fiber knob-derived peptides were produced, using 200 Ìg of peptide per animal and injection [19,24]. Both peptides induced antibodies in rabbits with titers in ELISA 1204,800 and 51,200, respectively, using the homologous peptide with 0.1 Ìg per well.…”