RESUMEN
The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.
Asunto(s)
Anticuerpos ampliamente neutralizantes/genética , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/genética , Linfocitos B/inmunología , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/inmunología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Epítopos , Femenino , Genotipo , Hepacivirus/genética , Hepatitis C/inmunología , Anticuerpos contra la Hepatitis C/química , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Masculino , Persona de Mediana Edad , Mutación , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
As exemplified by the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there is a strong demand for rapid high-throughput isolation pipelines to identify potent neutralizing antibodies for prevention and therapy of infectious diseases. However, despite substantial progress and extensive efforts, the identification and production of antigen-specific antibodies remains labor- and cost-intensive. We have advanced existing concepts to develop a highly efficient high-throughput protocol with proven application for the isolation of potent antigen-specific antibodies against human immunodeficiency virus 1, hepatitis C virus, human cytomegalovirus, Middle East respiratory syndrome coronavirus, SARS-CoV-2 and Ebola virus. It is based on computationally optimized multiplex primer sets (openPrimeR), which guarantee high coverage of even highly mutated immunoglobulin gene segments as well as on optimized antibody cloning and production strategies. Here, we provide the detailed protocol, which covers all critical steps from sample collection to antibody production within 12-14 d.