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. 2005 Jan 18;102(3):797-801.
doi: 10.1073/pnas.0409065102. Epub 2005 Jan 10.

Evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses

Zhi-yong Yang  1 , Heidi C Werner, Wing-pui Kong, Kwanyee Leung, Elisabetta Traggiai, Antonio Lanzavecchia, Gary J Nabel
Affiliations

Evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses

Zhi-yong Yang et al. Proc Natl Acad Sci U S A. .

Abstract

Molecular characterization of the severe acute respiratory syndrome coronavirus has revealed genetic diversity among isolates. The spike (S) glycoprotein, the major target for vaccine and immune therapy, shows up to 17 substitutions in its 1,255-aa sequence; however, the biologic significance of these changes is unknown. Here, the functional effects of S mutations have been determined by analyzing their affinity for a viral receptor, human angiotensin-converting enzyme 2 (hACE-2), and their sensitivity to Ab neutralization with viral pseudotypes. Although minor differences among eight strains transmitted during human outbreaks in early 2003 were found, substantial functional changes were detected in S derived from a case in late 2003 from Guangdong province [S(GD03T0013)] and from two palm civets, S(SZ3) and S(SZ16). S(GD03T0013) depended less on the hACE-2 receptor and was markedly resistant to Ab inhibition. Unexpectedly, Abs that neutralized most human S glycoproteins enhanced entry mediated by the civet virus S glycoproteins. The mechanism of enhancement involved the interaction of Abs with conformational epitopes in the hACE-2-binding domain. Finally, improved immunogens and mAbs that minimize this complication have been defined. These data show that the entry of severe acute respiratory syndrome coronaviruses can be enhanced by Abs, and they underscore the need to address the evolving diversity of this newly emerged virus for vaccines and immune therapies.

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Figures

Fig. 1.
Fig. 1.
Analysis of different S strains for sensitivity to Ab neutralization. (a) Lentivirus vectors prepared as described in refs. and were pseudotyped with the indicated SARS-CoV S glycoproteins from human isolates. These pseudoviruses were incubated with purified IgG from mice vaccinated with a DNA expression vector encoding S(Urbani) or negative control sera from mock-immunized mice. The inhibition by the negative control at each data point was subtracted from that of immune IgG. The percentage of inhibition assessed by luciferase reporter gene expression was calculated by the reduction in luciferase activity relative to values achieved in the absence of sera. Inhibition seen with control IgG was <10% in general compared with samples lacking IgG. (b) Ab-dependent enhancement of S entry in pseudoviruses from two palm civet pseudoviruses. (c) Pseudovirus from Urbani [S (Urbani)], resistant human [S(GD03T0013)], and palm civets [S(SZ3) and S(SZ16)] were incubated with human neutralizing mAbs S3.1, S127, and S111, which were derived from EBV-transformed B lymphocytes (15).
Fig. 2.
Fig. 2.
Differential sensitivity of S strains to ACE-2 inhibition and relative resistance of GD03T0013 and SZ3 to homologous neutralization. (a) The indicated pseudoviruses were incubated with increasing amounts of soluble recombinant hACE-2 or hACE (negative control, purified by similar methods) and entry was assessed by using the luciferase reporter gene as described for Fig. 1. (b) Purified IgG from animals immunized with S(GD03T0013) DNA expression vectors inhibits S(Urbani) but does not neutralize S(GD03T0013), S(SZ3), or S(SZ16) pseudoviruses. The pseudotyped viruses are shown according to their species origin (human or civet), shown above each graph. (c) Purified IgG from mice immunized with full-length S(SZ3) DNA expression vectors confers neutralization to S(Urbani) and partial inhibition of S(GD03T0013) but does not neutralize S(SZ3).
Fig. 3.
Fig. 3.
Definition of genetic determinants of S glycoprotein inhibition and enhancement and specific biochemical interaction of Abs with native S(Urbani) and S(SZ3). (a) Schematic diagram of sensitive S(Urbani), resistant palm civet S(SZ3), and chimeric glycoproteins SU and US as shown. (b) (Left) Gene transfer efficiency of pseudoviruses containing the specified wild-type and chimeric S in 786-O cells. Entry of S(Urbani) pseudovirus into 786-O cells is 100-fold more efficient than S(SZ3) pseudovirus. The dashed line indicates the background levels of gene transfer in the absence of S, SU, or US, as defined in a. (Right) Inhibition or enhancement of the indicated S pseudotypes with immune IgG to S(Urbani) shows the dependence on the hACE-2-binding domain. (c) (Right) Interaction of S(Urbani) and S(SZ3) with purified IgG from mice immunized with S(Urbani) full length (FL) gene was determined by immunoprecipitation (IP). (Left) The total amount of S protein used in immunoprecipitation was determined by Western blotting (Input).
Fig. 4.
Fig. 4.
Identification of immunogens and mAbs that circumvent Ab-dependent enhancement of entry and biochemical analysis of the mechanism of enhancement. (a) Neutralization profile of purified IgG from mice vaccinated with a DNA expression vector encoding a secreted form of S(Urbani) terminated at amino acid 1153, S(1153), compared against the indicated human or civet pseudoviruses. (b) (Right) Biochemical interaction of purified IgG from S(1153) DNA-immunized mice with native, expressed S(Urbani) and S(SZ3) proteins by immunoprecipitation (IP) with S(1153) IgG. (Left) The total amount of S protein in the same volume of cell lysates was assessed by Western blotting (Input). Mice were immunized with a S(Urbani) expression vector terminated at amino acid 1153. (c) mAb S110 inhibits S(Urbani) and does not enhance civet pseudovirus entry. S110 or an isotype control Ab (control) was incubated with the indicated pseudoviruses, and inhibition was assessed as described for Fig. 1. The species origin of the S for pseudovirus is shown above each panel (human or civet) in a and c.
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