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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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Characterization of Ab 256.A, Neutralization of pseudoviral infection by 256-IgG1. An anti-CXCR4 Ab 33-IgG1[42] was used as a negative control, 80R and 11A were used as positive control for Tor2- and GD03-viruses, respectively. B, Kinetic characterization of the binding of spike RBDs to 256-IgG1. Binding kinetics was evaluated similarly as described in Fig. 2. B. C, Competition of 256 for the binding of Tor2- or GD03-RBD-C9 to 293T-ACE2 cells. Left, competition for the binding of Tor2-RBD-Ig to 293T-ACE2 cells. 0.5 ug/mL of Tor2-RBD-Ig or control-Ig (Filled purple) used for the staining of 293T-ACE2 cells and the scFvs (control or 256) were used at 5 ug/mL to compete for the binding. Right, competition for the binding of GD03-RBD to 293T-ACE2 cells. The assay was the same as Fig. 2C except 256 was used here. D, Ab 256 Competition ELISA Assay. A fixed amount of 256 scFv expressing phages (256-phages) were mixed with various scFv-Fc antibody or full-length IgG1s at indicated antibody concentration, and the mixtures were then added to Tor2-RBD (left) or GD03-RBD (right) coated ELISA plate. The competition of 256-IgG1s for the binding of 256-phages to RBDs were determined by measuring the remaining binding of 256-phages using HRP-anti-M13 antibody. 256-phages homologous to 256-IgG1 were used as positive controls and both showed competition for 256-phage binding to Tor2-RBD and GD03-RBD, 80R or 11A did not show inhibition of 256-phages binding to either Tor2- or GD03-RBDs.
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ppat-1000197-g003: Characterization of Ab 256.A, Neutralization of pseudoviral infection by 256-IgG1. An anti-CXCR4 Ab 33-IgG1[42] was used as a negative control, 80R and 11A were used as positive control for Tor2- and GD03-viruses, respectively. B, Kinetic characterization of the binding of spike RBDs to 256-IgG1. Binding kinetics was evaluated similarly as described in Fig. 2. B. C, Competition of 256 for the binding of Tor2- or GD03-RBD-C9 to 293T-ACE2 cells. Left, competition for the binding of Tor2-RBD-Ig to 293T-ACE2 cells. 0.5 ug/mL of Tor2-RBD-Ig or control-Ig (Filled purple) used for the staining of 293T-ACE2 cells and the scFvs (control or 256) were used at 5 ug/mL to compete for the binding. Right, competition for the binding of GD03-RBD to 293T-ACE2 cells. The assay was the same as Fig. 2C except 256 was used here. D, Ab 256 Competition ELISA Assay. A fixed amount of 256 scFv expressing phages (256-phages) were mixed with various scFv-Fc antibody or full-length IgG1s at indicated antibody concentration, and the mixtures were then added to Tor2-RBD (left) or GD03-RBD (right) coated ELISA plate. The competition of 256-IgG1s for the binding of 256-phages to RBDs were determined by measuring the remaining binding of 256-phages using HRP-anti-M13 antibody. 256-phages homologous to 256-IgG1 were used as positive controls and both showed competition for 256-phage binding to Tor2-RBD and GD03-RBD, 80R or 11A did not show inhibition of 256-phages binding to either Tor2- or GD03-RBDs.

Mentions: In a parallel effort D480A- or D480G-Tor2 RBD coated on immmunotubes and conjugated to magnetic beads were used as panning targets together with a third non-immune scFv library to isolate the desired BnAbs. Only panning against D480A-magnetic beads resulted in D480A-RBD specific ELISA positive clones and only one clone, scFv 256 bound to cell-surface expressed D480A full-length S protein and showed neutralization activity against D480A S pseudotyped viruses (data not shown). 256-scFv was converted to whole human IgG1 and then tested separately for neutralization of Tor2-, GD03-, D480A-, and D480G-pseudotyped viruses. As shown in Fig. 3A, 256-IgG1 neutralized D480A and D480G (D480A>D480G) but with <80% activity at the highest Ab concentration tested (50 µg/mL). It also inhibited Tor2 viral entry but was much less potent than 80R and only marginally neutralized GD03. Furthermore, 256-IgG1 showed even lower neutralization titer in a micro-neutralization assay (Table 3) using Urbani (Tor2 equivalent) wild type and 80R escape viruses which may be explained, at least in part, by the conformational differences between the recombinant S protein and the S on the pseudotype or natural virus. Interestingly, kinetic studies showed that 256-IgG1 had high binding affinity with all three RBDs: Tor2-, GD03- and D480A-RBD (Fig. 3B). While this discrepancy between high binding affinity of Ab 256 and weak pseudo/micro neutralization activity could also be due to conformation difference of S, alternatively it could be based on 256's mechanism of neutralization which is not by direct competition of S binding to ACE2. As shown in Fig. 3C, Ab 256 does not compete with Tor2-RBD for cell-surface ACE2 receptor binding (left panel), but dramatically augmented GD03-RBD's binding with ACE2 (right panel). As expected the 256 epitope is also distinct from 80R and 11A, neither 80R nor 11A competed with 256 for Tor2- (Fig. 3D, left) or GD03-RBD binding (Fig. 3-D, right) in a competition ELISA assay. Thus, BnAbs against 80R escape mutant D480A/G as well as 2002/03 and 2003/04 strains were not isolated through numerous de novo variant S protein pannings using non-immune Ab-phage libraries.


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)

Characterization of Ab 256.A, Neutralization of pseudoviral infection by 256-IgG1. An anti-CXCR4 Ab 33-IgG1[42] was used as a negative control, 80R and 11A were used as positive control for Tor2- and GD03-viruses, respectively. B, Kinetic characterization of the binding of spike RBDs to 256-IgG1. Binding kinetics was evaluated similarly as described in Fig. 2. B. C, Competition of 256 for the binding of Tor2- or GD03-RBD-C9 to 293T-ACE2 cells. Left, competition for the binding of Tor2-RBD-Ig to 293T-ACE2 cells. 0.5 ug/mL of Tor2-RBD-Ig or control-Ig (Filled purple) used for the staining of 293T-ACE2 cells and the scFvs (control or 256) were used at 5 ug/mL to compete for the binding. Right, competition for the binding of GD03-RBD to 293T-ACE2 cells. The assay was the same as Fig. 2C except 256 was used here. D, Ab 256 Competition ELISA Assay. A fixed amount of 256 scFv expressing phages (256-phages) were mixed with various scFv-Fc antibody or full-length IgG1s at indicated antibody concentration, and the mixtures were then added to Tor2-RBD (left) or GD03-RBD (right) coated ELISA plate. The competition of 256-IgG1s for the binding of 256-phages to RBDs were determined by measuring the remaining binding of 256-phages using HRP-anti-M13 antibody. 256-phages homologous to 256-IgG1 were used as positive controls and both showed competition for 256-phage binding to Tor2-RBD and GD03-RBD, 80R or 11A did not show inhibition of 256-phages binding to either Tor2- or GD03-RBDs.
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ppat-1000197-g003: Characterization of Ab 256.A, Neutralization of pseudoviral infection by 256-IgG1. An anti-CXCR4 Ab 33-IgG1[42] was used as a negative control, 80R and 11A were used as positive control for Tor2- and GD03-viruses, respectively. B, Kinetic characterization of the binding of spike RBDs to 256-IgG1. Binding kinetics was evaluated similarly as described in Fig. 2. B. C, Competition of 256 for the binding of Tor2- or GD03-RBD-C9 to 293T-ACE2 cells. Left, competition for the binding of Tor2-RBD-Ig to 293T-ACE2 cells. 0.5 ug/mL of Tor2-RBD-Ig or control-Ig (Filled purple) used for the staining of 293T-ACE2 cells and the scFvs (control or 256) were used at 5 ug/mL to compete for the binding. Right, competition for the binding of GD03-RBD to 293T-ACE2 cells. The assay was the same as Fig. 2C except 256 was used here. D, Ab 256 Competition ELISA Assay. A fixed amount of 256 scFv expressing phages (256-phages) were mixed with various scFv-Fc antibody or full-length IgG1s at indicated antibody concentration, and the mixtures were then added to Tor2-RBD (left) or GD03-RBD (right) coated ELISA plate. The competition of 256-IgG1s for the binding of 256-phages to RBDs were determined by measuring the remaining binding of 256-phages using HRP-anti-M13 antibody. 256-phages homologous to 256-IgG1 were used as positive controls and both showed competition for 256-phage binding to Tor2-RBD and GD03-RBD, 80R or 11A did not show inhibition of 256-phages binding to either Tor2- or GD03-RBDs.
Mentions: In a parallel effort D480A- or D480G-Tor2 RBD coated on immmunotubes and conjugated to magnetic beads were used as panning targets together with a third non-immune scFv library to isolate the desired BnAbs. Only panning against D480A-magnetic beads resulted in D480A-RBD specific ELISA positive clones and only one clone, scFv 256 bound to cell-surface expressed D480A full-length S protein and showed neutralization activity against D480A S pseudotyped viruses (data not shown). 256-scFv was converted to whole human IgG1 and then tested separately for neutralization of Tor2-, GD03-, D480A-, and D480G-pseudotyped viruses. As shown in Fig. 3A, 256-IgG1 neutralized D480A and D480G (D480A>D480G) but with <80% activity at the highest Ab concentration tested (50 µg/mL). It also inhibited Tor2 viral entry but was much less potent than 80R and only marginally neutralized GD03. Furthermore, 256-IgG1 showed even lower neutralization titer in a micro-neutralization assay (Table 3) using Urbani (Tor2 equivalent) wild type and 80R escape viruses which may be explained, at least in part, by the conformational differences between the recombinant S protein and the S on the pseudotype or natural virus. Interestingly, kinetic studies showed that 256-IgG1 had high binding affinity with all three RBDs: Tor2-, GD03- and D480A-RBD (Fig. 3B). While this discrepancy between high binding affinity of Ab 256 and weak pseudo/micro neutralization activity could also be due to conformation difference of S, alternatively it could be based on 256's mechanism of neutralization which is not by direct competition of S binding to ACE2. As shown in Fig. 3C, Ab 256 does not compete with Tor2-RBD for cell-surface ACE2 receptor binding (left panel), but dramatically augmented GD03-RBD's binding with ACE2 (right panel). As expected the 256 epitope is also distinct from 80R and 11A, neither 80R nor 11A competed with 256 for Tor2- (Fig. 3D, left) or GD03-RBD binding (Fig. 3-D, right) in a competition ELISA assay. Thus, BnAbs against 80R escape mutant D480A/G as well as 2002/03 and 2003/04 strains were not isolated through numerous de novo variant S protein pannings using non-immune Ab-phage libraries.

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