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

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.
Author Summary The SARS-CoV caused a worldwide epidemic of SARS in 2002/03 and was responsible for this zoonotic infectious disease. The role of neutralizing antibody (nAb) mediated immune pressure in the evolution of SARS-CoV during the 2002/03 outbreak and a second 2003/04 zoonotic transmission is unknown. Here we demonstrate nAb responses elicited during natural infection clearly have strain-specific components which could have been the driving force for virus evolution in spike protein during intra-species transmission. In vitro immune pressure using 2002/03 strain-specific nAb 80R recapitulate a dominant escape mutation that was present in all 2003/04 animal and human viruses. We investigated how to generate a single broad nAb (BnAb) with activity against various natural viral variants of the 2002/03 and 2003/04 outbreaks as well as nAb escape mutants. Remarkably, amino acid changes in an activation-induced cytidine deaminase (AID) “hot spot” of somatic hypermutation and localized to a single VL CDR were successful in generating BnAbs. These results provide an effective strategy for generating BnAbs that should be generally useful for improving immune based anti-viral therapies as well as providing a foundation to directly manipulate virus evolution by blocking escape pathways.