Evaluation of human monoclonal antibody 80R for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants - PubMed (original) (raw)

Evaluation of human monoclonal antibody 80R for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants

Jianhua Sui et al. J Virol. 2005 May.

Abstract

In this report, the antiviral activity of 80R immunoglobulin G1 (IgG1), a human monoclonal antibody against severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein that acts as a viral entry inhibitor in vitro, was investigated in vivo in a mouse model. When 80R IgG1 was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced by more than 4 orders of magnitude to below assay limits. The essential core region of S protein required for 80R binding was identified as a conformationally sensitive fragment (residues 324 to 503) that overlaps the receptor ACE2-binding domain. Amino acids critical for 80R binding were identified. In addition, the effects of various 80R-binding domain amino acid substitutions which occur in SARS-like-CoV from civet cats, and which evolved during the 2002/2003 outbreak and in a 2003/2004 Guangdong index patient, were analyzed. The results demonstrated that the vast majority of SARS-CoVs are sensitive to 80R. We propose that by establishing the susceptibility and resistance profiles of newly emerging SARS-CoVs through early S1 genotyping of the core 180-amino-acid neutralizing epitope of 80R, an effective immunoprophylaxis strategy with 80R should be possible in an outbreak setting. Our study also cautions that for any prophylaxis strategy based on neutralizing antibody responses, whether by passive or active immunization, a genotyping monitor will be necessary for effective use.

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Figures

FIG. 1.

FIG. 1.

Truncations and point mutations of S1(318-510) were analyzed to define the 80R antibody epitope. S1 residues 318 to 510 fused to the Fc region of human IgG1 and truncation or mutation variants of S1(318-510) containing the indicated residues were metabolically labeled and precipitated by protein A or 80R scFv. (a) S1(324-503) was the smallest fragment bound to 80R. Either N-terminal or C-terminal truncation variants slightly smaller than that either had decreased expression or lost binding activity to 80R. (b) Critical residues for the 80R epitope were observed. Individual alanine substitution of glutamic acid 452 or aspartic acids 454 and 480 in the S1(318-510) fragment impaired or abolished binding to 80R. IP, immunoprecipitation.

FIG. 2.

FIG. 2.

Effects on 80R binding of variant amino acid substitutions of S protein that occur in animal SARS-like-CoVs and human SARS-CoVs. (a) Indicated amino acid residues in S1(318-510) of Tor2 were individually replaced with corresponding variant amino acids found in SARS-like-CoVs or other human SARS-CoVs. These residues also were replaced with alanine. Alterations of K344A, N479A, and T487A affected the binding to 80R to some degree. N479K substitution resulted in an ∼50% decrease in 80R binding, and D480G substitution totally abolished binding to 80R. (b) Multiple substitutions with the amino acids of civet SZ3 virus (344R/360S/479K/487S) in the S1(318-510)-Ig construct of Tor2 had no effect on 80R binding, as well as full-length S1(12-672) of SZ3, which was de novo synthesized. Multiple substitutions with the amino acids of human GD03T virus (344R/360S/472P/480G/487S) in the S1(318-510)-Ig construct of Tor2 and full-length S1(12-672) of GD03T completely lost binding to 80R scFv. (c) Full-length S protein of Tor2 and variants containing amino acid substitutions of isolates SZ3 or GD03T were precipitated by 1D4, which recognizes a C9 tag present at the carboxyl terminus of each S protein, or by 80R IgG1 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The binding activities of these full-length S proteins to 80R IgG1 were consistent with those of their receptor-binding domains (318 to 510) or S1 domains (12 to 672) to 80R scFv. WT, wild type; IP, immunoprecipitation.

FIG. 3.

FIG. 3.

80R IgG1 neutralization of pseudoviral infection mediated by full-length SARS-CoV spike variants. HIVs pseudotyped with the S protein from Tor2, SZ3, or GD03T isolates were incubated with the indicated concentration of 80R IgG1 (solid line, diamonds) or nonrelevant human IgG1 (solid line, squares) for 1 h prior to infection. At 48 h after infection, luciferase activities in target cells were measured and relative viral inhibition was calculated as the ratio of luciferase activity in the presence to absence of 80R IgG1 or nonrelevant human IgG1. (a) 80R IgG1 efficiently blocked Tor2 S protein-pseudotyped HIV infection, with a 90% inhibitory concentration around 2 μg/ml. (b) 80R IgG1 also efficiently neutralized SZ3 S protein pseudoviral infection. (c) In contrast to Tor2 and SZ3, GD03T S protein-pseudotyped virus was essentially resistant to the neutralization of 80R IgG1 with a concentration up to 50 μg/ml. These results are representative of two experiments with similar results.

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