Engineering topics
Publications and source records attributed to Wilson, Ian A. (ORCID:0000000264692419).
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Abstract Histidine phosphorylation is a non-canonical post-translational modification (PTM), with 1-phosphohistidine (1-pHis) and 3-phosphohistidine (3-pHis) isoforms, that is understudied due to a lack of robust reagents, including high-affinity pHis-specific antibodies. Engineering pHis antibodies is challenging due to the labile nature of its phosphoramidate (P-N) bond. We developed a strategy for in vitro engineering of antibodies for the detection of native 3-pHis targets, in which the rabbit SC44-8 anti-3-pTza mAb is humanized into a scaffold (hSC44) that is suitable for phage display. Six unique Fab phage-displayed hSC44 scaffold libraries were screened for antibodies that bound 3-pHis with higher affinity and had specificity for 3-pHis versus 3-pTza. hSC44.20N32F L , the best engineered antibody, has ~10-fold higher affinity for 3-pHis than parental hSC44. Eleven new Fab structures, including the first antibody-pHis peptide structures, together with structural and quantum mechanical calculations, provided molecular insights into 3-pHis and 3-pTza discrimination by hSC44.20N32F L and the increased affinity obtained through engineering. We demonstrated the utility of these high-affinity 3-pHis-specific antibodies for the recognition of pHis proteins in mammalian cells by immunoblotting and immunofluorescence staining. Our work describes a general method for engineering labile PTM-specific antibodies and provides novel antibodies for investigating the role of 3-pHis in cell biology.
A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.
Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.