Structural Insights into the Conformational Plasticity and Antibody Recognition of HIV-1 Env

Citation

Dam, Kim-Marie Anh (2023) Structural Insights into the Conformational Plasticity and Antibody Recognition of HIV-1 Env. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/8pvb-dx31. https://resolver.caltech.edu/CaltechTHESIS:06022023-003659646

Abstract

Acquired immunodeficiency syndrome (AIDS) and its causal agent, the human immunodeficiency virus 1 (HIV-1), remain a global public health concern since they were first identified in the early 1980s. Diligent research and gradual scientific advances have led to innovative strategies in HIV-1/AIDS prevention and treatment, transforming an obscure and deadly disease into a manageable condition with a normal life expectancy. Despite this progress, researchers have yet to develop a safe and effective vaccine against HIV-1. The work presented here describes a structural perspective related to the HIV-1 Envelope (Env) glycoprotein, the sole viral target of vaccines that seek to elicit neutralizing antibodies.

Env is the only viral protein on the surface of HIV-1 virions and is composed of a homotrimer of gp120/gp41 heterodimers. Env mediates entry into target cells by engaging the host receptor, CD4. CD4 triggers conformational changes in gp120, thereby enabling coreceptor recognition. Interactions with the host coreceptor trigger structural rearrangements in gp41 that facilitate fusion of host and viral membranes leading to infection. Our work builds upon our understanding of Env structural plasticity. First, we evaluated the conformational plasticity of soluble Env constructs using double electron-electron resonance (DEER) spectroscopy. This method measured distances between probes in Env subunits, allowing us to interpret the distribution in distances as Env flexibility. Our findings captured previously unseen nuances in static Env structures including gp41 elasticity and conformational heterogeneity associated with CD4-receptor binding. Although our work gave a new perspective on Env flexibility, it largely corroborated observations from static Env structures. Importantly, this suggested that soluble versions of Env, which serve as templates for immunogen design, retain favorable structural properties.

Informed with these insights in Env structure, we then sought to address a prevailing question related to receptor engagement: how many CD4 receptors are needed to induce gp120 conformation changes that lead to coreceptor binding followed by fusion? Prior work only characterized CD4-induced Env structural changes in Envs complexed with three soluble CD4 proteins. In our work, we designed and structurally characterized Envs bound to only one or two CD4 receptors. We found that Env engagement of one CD4 resulted in minor changes to the prefusion, closed Env conformation while Env bound to two CD4 molecules led to CD4-induced opening in the CD4-bound gp120s and a partially open conformation in the unliganded gp120.

Structural biology has also been leveraged to characterize the mechanism by which broadly neutralizing antibodies (bNAbs) recognize HIV-1 Env. We include an extensive review of how structural observations from antibodies bound to viral proteins contribute to our understanding of antibody-mediated viral neutralization. We also present a technical evaluation of bNAb binding assays that revealed how Env conformations can be unintentionally altered resulting in misleading antibody binding results and identified ideal methods to ensure reliable data.

Additionally, we report on projects related to bNAbs that target the CD4 binding site (CD4bs) epitope of Env. In the first, we characterized the inferred germline (iGL) precursor of BG24, a VRC01-class bNAb with features that make it a promising target for vaccine design. We solved four cryo-EM structures of BG24iGL constructs complexed with different Envs and provided insight on the mode of iGL accommodation. The second project centers around the IOMA-class of CD4bs bNAbs. We characterized features of IOMA-class bNAbs and measured how different features contribute to neutralization breadth and potency. Taken together, the conclusions from our work provide guidance for the next generation of structure-based, CD4bs-targeting immunogen design.

Thesis Files