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.