A single amino acid substitution in the S1 and S2 Spike protein domains determines the neutralization escape phenotype of SARS-CoV - PubMed (original) (raw)

A single amino acid substitution in the S1 and S2 Spike protein domains determines the neutralization escape phenotype of SARS-CoV

Yu-ya Mitsuki et al. Microbes Infect. 2008 Jul.

Abstract

In response to SARS-CoV infection, neutralizing antibodies are generated against the Spike (S) protein. Determination of the active regions that allow viral escape from neutralization would enable the use of these antibodies for future passive immunotherapy. We immunized mice with UV-inactivated SARS-CoV to generate three anti-S monoclonal antibodies, and established several neutralization escape mutants with S protein. We identified several amino acid substitutions, including Y442F and V601G in the S1 domain and D757N and A834V in the S2 region. In the presence of each neutralizing antibody, double mutants with substitutions in both domains exhibited a greater growth advantage than those with only one substitution. Importantly, combining two monoclonal antibodies that target different epitopes effected almost complete suppression of wild type virus replication. Thus, for effective passive immunotherapy, it is important to use neutralizing antibodies that recognize both the S1 and S2 regions.

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Figures

Fig. 1

Fig. 1

The neutralizing activity of mAbs. Wild type virus were incubated for 1 h in the absence or presence of serially diluted control IgG1 or anti-S mAbs, and then used to inoculate Vero E6 cells. Infected cells were overplayed with 1% low melting agarose and cultured for 3 days. The number of plaques obtained in the presence of mAb was calculated as a percentage relative to those obtained in the presence of control IgG1. The means of two independent experiments are shown with standard deviation (SD).

Fig. 2

Fig. 2

Characterization of mAbs. (A) Immunofluorescence detection of SARS-CoV S protein. SARS-CoV-infected or uninfected Vero E6 cells were cultured for 40 h. Prior to development of an extensive CPE, cells were fixed, permeabilized and incubated with anti-S mAbs SKOT3, SKOT19 or SKOT20, followed by incubation with FITC-conjugated anti-mouse IgG. Nuclei were counter-stained with DAPI. The slides were examined using confocal microscopy. (B) Flow cytometric detection of SARS-CoV S protein. S6.2 cells expressing S protein were stained with biotinylated anti-S mAbs using one antibody (blue line), a combination (blue line), or control IgG (gray line), followed by incubation with streptavidin-APC. Stained cells were analyzed by FACScalibur: Upper panel: single staining patterns of SKOT3, SKOT19 and SKOT20. Lower panel: mixted staining patterns of SKOT3 and SKOT19 (left panel), or SKOT19 and SKOT20 (right panel). Control staining was depicted as shadows. (C) Competition assay of mAbs. The antibody competition assay was conducted with the combinations of unlabeled monoclonal antibodies (SKOT3, SKOT19, SKOT20) and biotinylated antibodies by ELISA using a plate coated with UV-inactivated SARS-CoV virion. The means of four independent experiments are shown with SD.

Fig. 3

Fig. 3

The neutralization resistant phenotype of escape mutant clones. Wild type and plaque-purified virus clones were incubated for 1 h in the presence of serially diluted anti-S mAbs or control IgG1, and then used to inoculate Vero E6 cells. Infected cells were overplayed with 1% low melting agarose and cultured for 3 days. The percentage of plaques obtained in the presence of mAb was determined relative to those obtained in the absence of selection mAb. The plaque count was carried out in duplicate and means of two wells are shown. (A) Comparison between wild type and v3 clones (left panel), v19 clones (middle panel) or v20 clones (right panel). The experiment was repeated with consistent results. (B) Wild type and plaque-purified virus clones were incubated for 1 h in the presence of serially diluted anti-S mAbs or control IgG1 that were different from the mAb used for selection. V3-1, v19-5 and v20-4 were neutralized by SKOT3 (left panel), SKOT19 (middle panel) or SKOT20 (right panel). A titration curve of a wild type virus was depicted with a dotted line. The means of two independent experiments are shown with SD.

Fig. 4

Fig. 4

Complete neutralization by a combination of any two mAbs. Wild type virus was incubated with or without SKOT3 (A = 1.0, B = 2.0, C = 4.0 μg/ml), SKOT19 (A = 0.05, B = 0.1, C = 0.2 μg/ml), SKOT20 (A = 0.025, B = 0.05, C = 0.1 μg/ml) or a mixture of two mAbs. Following infection of Vero E6 cells, the plaques were counted and the percent reduction calculated. The means of two independent experiments are shown with SD.

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