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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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Sequence alignment of the Vk of five Abs identified from 80R-Vk-cs library and 80R.The best-matched germline Vκ and Jκ genes of 80R are showed on top of the alignment. CDR regions are labeled in large boxes, amino acid substitutions from germline are colored in blue. A dash indicates no amino acid at that position. Amino acids 161–164 in CDRL1 were highlighted in pink. All 5 Abs have one consensus change from S to N at position 163. WRCY hot spots of AID are colored in green.
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ppat-1000197-g004: Sequence alignment of the Vk of five Abs identified from 80R-Vk-cs library and 80R.The best-matched germline Vκ and Jκ genes of 80R are showed on top of the alignment. CDR regions are labeled in large boxes, amino acid substitutions from germline are colored in blue. A dash indicates no amino acid at that position. Amino acids 161–164 in CDRL1 were highlighted in pink. All 5 Abs have one consensus change from S to N at position 163. WRCY hot spots of AID are colored in green.

Mentions: Structural data guided a different approach to engineer 80R to have more broadly neutralizing activity against D480A/G and 2003/04 outbreak strains. The co-crystal structure of the Tor2-RBD in complex with 80R shows that D480 lies at the center of RBD-80R interface. In addition, all of the D480 contacting residues are located in the Vκ light chain. In particular in 80R CDRL1, D480 makes an intermolecular salt bridge to R162 that is flanked by two neutral residues: V161 and S163, and an H-bond to N164 [24]. Other contacting residues are D182 in CDRL2 and R223 in CDRL3. Notably, amino acids 162–164 form part of a WRCY “hot spot” motif for AID-mediated somatic hypermutation (SHM) [27],[28] and R162 and N164 are mutated from germline serine (Fig. 4). This suggested firstly that natural mutations within this hot spot would likely exist in our circa 108 member non-immune Vkappa (Vκ) repertoire and secondly, that focused mutagenesis on this “hot spot” would also provide an experimental system to test whether mutations within this region would broaden binding and neutralization activity. Accordingly two directed approaches, Vκ light chain shuffling (cs) and CDRL1 (amino acids 161–164) focused mutagenesis (fm) were simultaneously utilized to identify natural or directed variation in critical Vκ contact amino acids, respectively.


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)

Sequence alignment of the Vk of five Abs identified from 80R-Vk-cs library and 80R.The best-matched germline Vκ and Jκ genes of 80R are showed on top of the alignment. CDR regions are labeled in large boxes, amino acid substitutions from germline are colored in blue. A dash indicates no amino acid at that position. Amino acids 161–164 in CDRL1 were highlighted in pink. All 5 Abs have one consensus change from S to N at position 163. WRCY hot spots of AID are colored in green.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000197-g004: Sequence alignment of the Vk of five Abs identified from 80R-Vk-cs library and 80R.The best-matched germline Vκ and Jκ genes of 80R are showed on top of the alignment. CDR regions are labeled in large boxes, amino acid substitutions from germline are colored in blue. A dash indicates no amino acid at that position. Amino acids 161–164 in CDRL1 were highlighted in pink. All 5 Abs have one consensus change from S to N at position 163. WRCY hot spots of AID are colored in green.
Mentions: Structural data guided a different approach to engineer 80R to have more broadly neutralizing activity against D480A/G and 2003/04 outbreak strains. The co-crystal structure of the Tor2-RBD in complex with 80R shows that D480 lies at the center of RBD-80R interface. In addition, all of the D480 contacting residues are located in the Vκ light chain. In particular in 80R CDRL1, D480 makes an intermolecular salt bridge to R162 that is flanked by two neutral residues: V161 and S163, and an H-bond to N164 [24]. Other contacting residues are D182 in CDRL2 and R223 in CDRL3. Notably, amino acids 162–164 form part of a WRCY “hot spot” motif for AID-mediated somatic hypermutation (SHM) [27],[28] and R162 and N164 are mutated from germline serine (Fig. 4). This suggested firstly that natural mutations within this hot spot would likely exist in our circa 108 member non-immune Vkappa (Vκ) repertoire and secondly, that focused mutagenesis on this “hot spot” would also provide an experimental system to test whether mutations within this region would broaden binding and neutralization activity. Accordingly two directed approaches, Vκ light chain shuffling (cs) and CDRL1 (amino acids 161–164) focused mutagenesis (fm) were simultaneously utilized to identify natural or directed variation in critical Vκ contact amino acids, respectively.

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