BPS2026 – Optimizing cryo-EM sample preparation for HCMV gB-antibody complexes

Human cytomegalovirus (HCMV), part of the Herpesviridae family, infects 60%–90% of the global population for life. It is the leading infectious cause of neurological and developmental birth defects in newborns and poses serious risks to immunocompromised individuals—such as transplant recipients, chemotherapy patients, and people with HIV/AIDS, resulting in significant illness and death. Despite its impact, there is no FDA-approved vaccine, and current antiviral treatments can be toxic, making the development of safer and more effective therapies a high priority. Glycoprotein B (gB) is a key HCMV antigen and entry protein that acts as a membrane fusogen. Its essential role in viral entry makes it a promising therapeutic target. However, limited knowledge of gB-specific immune responses—especially neutralizing antibodies—has hindered progress in gB-based treatments. Our lab recently isolated a panel of novel neutralizing antibodies from HCMV-seropositive individuals using a stabilized, prefusion form of gB. Mapping their epitopes could enhance understanding of gB antigenicity and inform vaccine and therapeutic design. One major challenge is the metastability of prefusion gB, which complicates structural studies and epitope mapping during cryo-EM grid preparation. Moreover, each antibody interacts differently with gB, requiring tailored sample preparation. In this study, we optimized the preparation of gB-antibody complexes to maintain structural integrity during EM grid application and screening. We refined conditions for each antibody—including grid type, blotting parameters, and buffer composition—to preserve the complexes and reduce preferred particle orientations. These protocols offer a reliable approach for handling gB-antibody samples in structural studies.