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Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway.

Sui J, Aird DR, Tamin A, Murakami A, Yan M, Yammanuru A, Jing H, Kan B, Liu X, Zhu Q, Yuan QA, Adams GP, Bellini WJ, Xu J, Anderson LJ, Marasco WA - PLoS Pathog. (2008)

Bottom Line: Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection.Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs.These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA. Jianhua_sui@dfci.harvard.edu

ABSTRACT
Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.

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Neutralization of pseudotyped viral infection by anti-GD03 Abs and epitope mapping of GD03 nAb 11A.A, Five anti-GD03 Abs isolated by phage Ab library screening against purified GD03-RBD were analyzed for neutralizing activity using GD03 (Left panel) or Tor2 (Right panel) spike pseudoyped viruses. B, Kinetic characterization of GD03-RBD binding to 11A-IgG1. Abs were captured on a CM4 chip via immobilized anti-human IgG1 on the chip. GD03-RBD at various concentrations (2 folds serial dilutions, highest concentration was indicated) was injected over the chip surface. Binding kinetics was evaluated using a 1∶1 interaction model. In each panel, the binding response curves (red lines) are overlaid with the fit of the interaction model (black lines). All ka, kd, KD value showed in the table represent the means and standard errors of three experiments. C, Competition of 11A for the binding of GD03-RBD-C9 to 293T-ACE2 cells. GD03-RBD-C9 or control-RBD-C9 (Filled purple) used for staining is at 20 ug/mL and the Abs (256 or 80R negative control) were used at 50 ug/mL to compete for the binding of GD03-RBD to 293T-ACE2 cells. D, Epitope mapping of 11A. Purified proteins of a set of GD03-RBD or Tor2-RBD mutants were coated to ELISA plates at indicated concentrations. 2 µg/ml of 11A-IgG1 followed by HRP-anti-human IgG1 were used to detect the binding of 11A with different mutants. E, Interface of structure of the Tor2- RBD/80R complex 21. S1-RBD is in yellow. CDR loops (H1-H3 and L1-L3) of 80R, amino acids of 80R-CDRL1 and L2 (162–164 and 182) as well as 472L and 480D of S1-RBD are colored as shown.
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ppat-1000197-g002: Neutralization of pseudotyped viral infection by anti-GD03 Abs and epitope mapping of GD03 nAb 11A.A, Five anti-GD03 Abs isolated by phage Ab library screening against purified GD03-RBD were analyzed for neutralizing activity using GD03 (Left panel) or Tor2 (Right panel) spike pseudoyped viruses. B, Kinetic characterization of GD03-RBD binding to 11A-IgG1. Abs were captured on a CM4 chip via immobilized anti-human IgG1 on the chip. GD03-RBD at various concentrations (2 folds serial dilutions, highest concentration was indicated) was injected over the chip surface. Binding kinetics was evaluated using a 1∶1 interaction model. In each panel, the binding response curves (red lines) are overlaid with the fit of the interaction model (black lines). All ka, kd, KD value showed in the table represent the means and standard errors of three experiments. C, Competition of 11A for the binding of GD03-RBD-C9 to 293T-ACE2 cells. GD03-RBD-C9 or control-RBD-C9 (Filled purple) used for staining is at 20 ug/mL and the Abs (256 or 80R negative control) were used at 50 ug/mL to compete for the binding of GD03-RBD to 293T-ACE2 cells. D, Epitope mapping of 11A. Purified proteins of a set of GD03-RBD or Tor2-RBD mutants were coated to ELISA plates at indicated concentrations. 2 µg/ml of 11A-IgG1 followed by HRP-anti-human IgG1 were used to detect the binding of 11A with different mutants. E, Interface of structure of the Tor2- RBD/80R complex 21. S1-RBD is in yellow. CDR loops (H1-H3 and L1-L3) of 80R, amino acids of 80R-CDRL1 and L2 (162–164 and 182) as well as 472L and 480D of S1-RBD are colored as shown.

Mentions: GD03-RBD was initially used as bait to isolate cross-strain nAbs that could recognize promiscuous amino acids at positions 472 and 480. Five unique antibodies (11A, 10C, 15D, 23E, 28G) were identified by panning against GD03-RBD-C9 with two non-immune human Ab phage display libraries and neutralizing activity was tested against pseudotyped viruses. One of them 11A was found to be a potent nAb against GD03 but not against Tor2 (Fig. 2A). Kinetic analysis of 11A IgG binding to GD03-RBD demonstrated high affinity interaction (KD = 2.2±0.7 nM) (Fig. 2B). 11A also potently inhibited GD03-RBD's binding to ACE2 expressing 293T cells (Fig. 2C). Epitope mapping showed that two amino acids in the GD03-RBD region (472P and 480G) were critically important for 11A's binding. Single amino acid changes at either position resulted in complete loss of 11A binding (Fig. 2D). Other amino acid changes at positions of V404A, Y442F, K465A, T468A and S487A did not affect 11A binding (data not shown). On the basis of this result, all of the 2004 civet viruses (PC04) should also be recognized and neutralized by 11A since PC04 has same 472P and 480G as GD03 (Table 2). In contrast to 11A, 80R was only affected by changes at position 480 but not 472 [23]. As shown in Fig. 2E, 472 is located on a ridge formed by an extended loop that is reinforced by the Cys467-Cys474 disulfide bond [26]. This suggests that 11A recognizes a slightly different neutralizing epitope since the 80R CDR residues do not make contact with this segment of RBD [24].


Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway.

Sui J, Aird DR, Tamin A, Murakami A, Yan M, Yammanuru A, Jing H, Kan B, Liu X, Zhu Q, Yuan QA, Adams GP, Bellini WJ, Xu J, Anderson LJ, Marasco WA - PLoS Pathog. (2008)

Neutralization of pseudotyped viral infection by anti-GD03 Abs and epitope mapping of GD03 nAb 11A.A, Five anti-GD03 Abs isolated by phage Ab library screening against purified GD03-RBD were analyzed for neutralizing activity using GD03 (Left panel) or Tor2 (Right panel) spike pseudoyped viruses. B, Kinetic characterization of GD03-RBD binding to 11A-IgG1. Abs were captured on a CM4 chip via immobilized anti-human IgG1 on the chip. GD03-RBD at various concentrations (2 folds serial dilutions, highest concentration was indicated) was injected over the chip surface. Binding kinetics was evaluated using a 1∶1 interaction model. In each panel, the binding response curves (red lines) are overlaid with the fit of the interaction model (black lines). All ka, kd, KD value showed in the table represent the means and standard errors of three experiments. C, Competition of 11A for the binding of GD03-RBD-C9 to 293T-ACE2 cells. GD03-RBD-C9 or control-RBD-C9 (Filled purple) used for staining is at 20 ug/mL and the Abs (256 or 80R negative control) were used at 50 ug/mL to compete for the binding of GD03-RBD to 293T-ACE2 cells. D, Epitope mapping of 11A. Purified proteins of a set of GD03-RBD or Tor2-RBD mutants were coated to ELISA plates at indicated concentrations. 2 µg/ml of 11A-IgG1 followed by HRP-anti-human IgG1 were used to detect the binding of 11A with different mutants. E, Interface of structure of the Tor2- RBD/80R complex 21. S1-RBD is in yellow. CDR loops (H1-H3 and L1-L3) of 80R, amino acids of 80R-CDRL1 and L2 (162–164 and 182) as well as 472L and 480D of S1-RBD are colored as shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2572002&req=5

ppat-1000197-g002: Neutralization of pseudotyped viral infection by anti-GD03 Abs and epitope mapping of GD03 nAb 11A.A, Five anti-GD03 Abs isolated by phage Ab library screening against purified GD03-RBD were analyzed for neutralizing activity using GD03 (Left panel) or Tor2 (Right panel) spike pseudoyped viruses. B, Kinetic characterization of GD03-RBD binding to 11A-IgG1. Abs were captured on a CM4 chip via immobilized anti-human IgG1 on the chip. GD03-RBD at various concentrations (2 folds serial dilutions, highest concentration was indicated) was injected over the chip surface. Binding kinetics was evaluated using a 1∶1 interaction model. In each panel, the binding response curves (red lines) are overlaid with the fit of the interaction model (black lines). All ka, kd, KD value showed in the table represent the means and standard errors of three experiments. C, Competition of 11A for the binding of GD03-RBD-C9 to 293T-ACE2 cells. GD03-RBD-C9 or control-RBD-C9 (Filled purple) used for staining is at 20 ug/mL and the Abs (256 or 80R negative control) were used at 50 ug/mL to compete for the binding of GD03-RBD to 293T-ACE2 cells. D, Epitope mapping of 11A. Purified proteins of a set of GD03-RBD or Tor2-RBD mutants were coated to ELISA plates at indicated concentrations. 2 µg/ml of 11A-IgG1 followed by HRP-anti-human IgG1 were used to detect the binding of 11A with different mutants. E, Interface of structure of the Tor2- RBD/80R complex 21. S1-RBD is in yellow. CDR loops (H1-H3 and L1-L3) of 80R, amino acids of 80R-CDRL1 and L2 (162–164 and 182) as well as 472L and 480D of S1-RBD are colored as shown.
Mentions: GD03-RBD was initially used as bait to isolate cross-strain nAbs that could recognize promiscuous amino acids at positions 472 and 480. Five unique antibodies (11A, 10C, 15D, 23E, 28G) were identified by panning against GD03-RBD-C9 with two non-immune human Ab phage display libraries and neutralizing activity was tested against pseudotyped viruses. One of them 11A was found to be a potent nAb against GD03 but not against Tor2 (Fig. 2A). Kinetic analysis of 11A IgG binding to GD03-RBD demonstrated high affinity interaction (KD = 2.2±0.7 nM) (Fig. 2B). 11A also potently inhibited GD03-RBD's binding to ACE2 expressing 293T cells (Fig. 2C). Epitope mapping showed that two amino acids in the GD03-RBD region (472P and 480G) were critically important for 11A's binding. Single amino acid changes at either position resulted in complete loss of 11A binding (Fig. 2D). Other amino acid changes at positions of V404A, Y442F, K465A, T468A and S487A did not affect 11A binding (data not shown). On the basis of this result, all of the 2004 civet viruses (PC04) should also be recognized and neutralized by 11A since PC04 has same 472P and 480G as GD03 (Table 2). In contrast to 11A, 80R was only affected by changes at position 480 but not 472 [23]. As shown in Fig. 2E, 472 is located on a ridge formed by an extended loop that is reinforced by the Cys467-Cys474 disulfide bond [26]. This suggests that 11A recognizes a slightly different neutralizing epitope since the 80R CDR residues do not make contact with this segment of RBD [24].

Bottom Line: Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection.Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs.These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA. Jianhua_sui@dfci.harvard.edu

ABSTRACT
Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.

Show MeSH
Related in: MedlinePlus