Reversion of Somatic Mutations of the Respiratory Syncytial Virus-Specific Human Monoclonal Antibody Fab19 Reveal a Direct Relationship between Association Rate and Neutralizing Potency
ABSTRACT The role of affinity in determining neutralizing potency of mAbs directed against viruses is not well understood. We investigated the kinetic, structural, and functional advantage conferred by individual naturally occurring somatic mutations in the Ab H chain V region of Fab19, a well-described neutralizing human mAb directed to respiratory syncytial virus. Comparison of the affinity-matured Ab Fab19 with recombinant Fab19 Abs that were variants containing reverted amino acids from the inferred unmutated ancestor sequence revealed the molecular basis for affinity maturation of this Ab. Enhanced binding was achieved through mutations in the third H chain CDR (HCDR3) that conferred a markedly faster on-rate and a desirable increase in antiviral neutralizing activity. In contrast, most somatic mutations in the HCDR1 and HCDR2 regions did not significantly enhance Ag binding or antiviral activity. We observed a direct relationship between the measured association rate (Kon) for F protein and antiviral activity. Modeling studies of the structure of the Ag-Ab complex suggested the HCDR3 loop interacts with the antigenic site A surface loop of the respiratory syncytial virus F protein, previously shown to contain the epitope for this Ab by experimentation. These studies define a direct relationship of affinity and neutralizing activity for a viral glycoprotein-specific human mAb.
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ABSTRACT: Neutralizing antibodies (nAbs) targeting glycoprotein E2 are important to control hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412-423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a β-hairpin in complex with three independent nAbs. Our structure of the same peptide in complex with nAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412-423 are essential for virus entry, but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and β-hairpin conformation, respectively) display similar neutralizing activity despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as immune evasion strategy contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and non-enveloped viruses. Approximately 180 million people worldwide are infected with Hepatitis C virus (HCV) and neutralizing antibodies play an important role in controlling replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412-423) - that is disordered in the recently reported crystal structure of an E2 core fragment - can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activity in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than to other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this conserved antigenic region contributes to the evasion of the humoral host immune response, facilitating chronicity and viral spread of HCV within an infected individual. Copyright © 2014, American Society for Microbiology. All Rights Reserved.Journal of Virology 12/2014; 89(4). DOI:10.1128/JVI.02190-14 · 4.65 Impact Factor
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ABSTRACT: To improve a previously constructed broadly neutralizing hepatitis B virus (HBV)-specific preS1 humanized antibody (HzKR127), we further humanized it through specificity-determining residue (SDR) grafting. Moreover, we improved affinity by mutating two residues in heavy-chain complementarity-determining regions (CDR), on the basis of the crystal structure of the antigen-antibody complex. HzKR127-3.2 exhibited 2.5-fold higher affinity and enhanced virus-neutralizing activity compared to the original KR127 antibody and showed less immunogenic potential than HzKR127. Enhanced virus-neutralizing activity was achieved by the increased association rate, providing insights into engineering potent antibody therapeutics for HBV immunoprophylaxis. HzKR127-3.2 may be a good candidate for HBV immunoprophylaxis. Copyright © 2014. Published by Elsevier B.V.FEBS Letters 12/2014; 589(2). DOI:10.1016/j.febslet.2014.11.046 · 3.34 Impact Factor
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ABSTRACT: The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies.PLoS Pathogens 09/2014; 10(9):e1004403. DOI:10.1371/journal.ppat.1004403 · 8.14 Impact Factor