Structural and antigenic characterization of human cytomegalovirus gB

PROJECT SUMMARY/ABSTRACT Human cytomegalovirus (HCMV) is a double-stranded-DNA, enveloped virus that infects most of the global population by establishing lifelong latent infections. It poses the greatest risk, however, to the immunocompromised and is the leading cause of congenital birth defects in the US and Europe. Much of its

success as a pathogen stem from its efficient entry mechanism and immune evasion tactics. To enter target cells, HCMV requires glycoprotein B (gB), the membrane fusogen that functions by refolding from the metastable prefusion to the stable postfusion form. While the involvement of gB in entry is clear, we do

not yet fully understand how each form contributes to the anti-HCMV host immune response. It is known that HCMV gB is a major target of the humoral immune response and elicits antibodies (Abs), but a vast majority of these Abs are non-neutralizing. Moreover, known anti-gB neutralizing (n) Abs bind both pre- and postfusion

forms. Such lack of specificity may render them less effective. However, studies in other viruses, such as respiratory syncytial virus, have identified prefusion-specific antibodies with strong neutralization and protection properties. Therefore, HCMV prefusion-gB-specific Abs may exist and have potential to be highly potent and

protective. However, isolation of these Abs has long been hindered by an inability to stabilize prefusion gB. We have engineered a soluble, prefusion-stabilized gB (s-gBpre) and I have verified that at a low resolution it structurally resembles native gBpre. Additionally, our collaborators have isolated new Abs specific to s-gBpre.

However, it is not clear whether s-gBpre recapitulates native gBpre epitopes and if these new Abs have neutralizing properties. Based on preliminary data, I hypothesize that s-gBpre is a promising reagent for the development of Ab-based anti-HCMV therapeutics because it represents the active form of the fusogen with epitopes ideally

presented for development of robust neutralizing and protective antibodies. In Aim 1, I will determine the high- resolution structure of s-gBpre by cryo-EM, alone and bound to known nAbs, to validate it and identify the epitopes with the highest antigenic potential. In Aim 2, I will isolate and characterize new anti-gBpre Abs and examine their

binding to s-gBpre. This proposal is innovative because it describes the first engineered s-gBpre to isolate new anti-HCMV nAbs. It is significant because long term, the epitope mapping of prefusion-gB will aid in developing a gB-based immunogen for the first HCMV vaccine, and to isolate neutralizing and protective anti-HCMV

monoclonal Abs. In addition to its potential to make major contributions to the development of anti-HCMV therapeutics, this proposal will provide me with extensive training towards my development into a premier scientist. My sponsor, Dr. Ekaterina Heldwein, and collaborator, Dr. Andrew McGuire, will ensure that throughout this project, I acquire

the necessary skills for the successful completion of the proposed work and, in the future, for my successful career as an independent investigator.