Nathaniel Felbinger

Many protein functions are coupled to proton transfer, yet the underlying mechanisms remain extremely difficult to resolve by experiment or conventional molecular dynamics (MD) simulations. The human voltage-gated proton channel H _V 1 (hH _V 1) plays key roles in human physiology, but how voltage and pH together drive its activation remains unresolved. Experimental structures of hH _V 1 are limited to the X-ray structure of a mouse chimera and NMR models of a truncated hH _V 1, both representing an intermediate resting state. Here we determine the activation pathway of hH _V 1, together with structural models of its resting and activated states, using an approach that integrates all-atom continuous constant-pH MD (CpHMD), weighted-ensemble (WE) path sampling, and AlphaFold modeling. Starting from an AlphaFold model selected for consistency with resting-state experimental data, over 100 µ s of simulations reveal a mechanism in which transmembrane voltage, assisted by proton titration, drives an outward displacement of the transmembrane segment S4 accompanied by a one-click reorganization of countercharge pairings. These events are enabled by a delicately balanced electrostatic landscape and pH-modulated breathing motions in the resting state that prime the channel for protonation-state switches upon voltage sensing, a principle that may underlie other proton-coupled voltage-gating processes. The simulations also uncover lipid-binding sites at which POPG selectively stabilizes the S4-up conformation, whereas POPE can stabilize either state. These predictions are validated against extensive electrophysiology, mutagenesis data, and new smFRET experiments, which support the one-click S4 displacement and the lipid effects. WE-CpHMD, implemented in the AMBER package, is applicable to other proton-coupled processes that remain elusive.

The development of an effective prophylactic hepatitis C virus (HCV) vaccine is a priority to achieve global elimination of the virus. Accurate assessment of the neutralizing breadth of antibodies induced by vaccines and a clear understanding of the antigenic differences between viral variants included in vaccines are both critical for vaccine development. Prior studies have indicated that HCV genotypes (gts) do not dictate the sensitivity of HCV envelope glycoprotein (E1E2) variants to neutralizing antibodies. However, most of these prior studies under-sampled variants from gts 2–6. Here, we selected a genetically diverse and representative panel of gt 2–6 E1E2 variants, used them to generate HCV pseudoparticles (HCVpp), and measured neutralization of these HCVpp by neutralizing antibodies and HCV-immune plasma from persons infected with gt 1–6 viruses. We found that neutralization results obtained with this gt 2–6 panel were remarkably similar to results obtained with a previously described, antigenically diverse, gt 1-predominant reference panel of 15 HCVpp. These data confirm that, even considering genetically diverse HCV variants across gt 1–6, E1E2 antigenicity is not dictated by gt, and that the previously published panel of 15 HCVpp represents neutralization of all HCV gts with reasonable accuracy.

Hepatitis C virus (HCV) is a major medical health burden and the leading cause of chronic liver disease and cancer worldwide. More than 58 million people are chronically infected with HCV, with 1.5 million new infections occurring each year. An effective HCV vaccine is a major public health and medical need as recognized by the World Health Organization. However, due to the high variability of the virus and its ability to escape the immune response, HCV rapidly accumulates mutations, making vaccine development a formidable challenge. An effective vaccine must elicit broadly neutralizing antibodies (bnAbs) in a consistent fashion. After decades of studies from basic research through clinical development, the antigen of choice is considered the E1E2 envelope glycoprotein due to conserved, broadly neutralizing antigenic domains located in the constituent subunits of E1, E2, and the E1E2 heterodimeric complex itself. The challenge has been elicitation of robust humoral and cellular responses leading to broad virus neutralization due to the relatively low immunogenicity of this antigen. In view of this challenge, structure-based vaccine design approaches to stabilize key antigenic domains have been hampered due to the lack of E1E2 atomic-level resolution structures to guide them. Another challenge has been the development of a delivery platform in which a multivalent form of the antigen can be presented in order to elicit a more robust anti-HCV immune response. Recent nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both the cellular and humoral components of the immune system. This review focuses on recent advances in understanding the E1E2 heterodimeric structure to facilitate a rational design approach and the potential for development of a multivalent nanoparticle-based HCV E1E2 vaccine. Both aspects are considered important in the development of an effective HCV vaccine that can effectively address viral diversity and escape.

Hepatitis C virus (HCV) is a major medical health burden and leading cause of chronic liver disease and cancer worldwide. More than 58 million people are chronically infected with HCV, with 1.5 million new infections occurring each year. An effective HCV vaccine is a major public health and medical need as recognized by the World Health Organization. However, due to the high variability of the virus and its ability to escape the immune response, HCV rapidly accumulates mutations making a vaccine a formidable challenge. An effective vaccine must elicit broadly neutralizing antibodies (bnAbs) in a consistent fashion. After decades of studies from basic research through clinical development, the antigen of choice is considered to be the E1E2 envelope glycoprotein due to conserved, broadly neutralizing antigenic domains located in the constituent subunits of E1, E2, and the E1E2 heterodimeric complex itself. The challenge has been elicitation of robust humoral and cellular responses leading to broad virus neutralization due to the relatively low immunogenicity of this antigen. In view of this challenge, structure-based vaccine design approaches to stabilize key antigenic domains have been hampered due to the lack of E1E2 atomic-level resolution structures to guide them. Another challenge has been development of a delivery platform in which a multivalent form of the antigen can be presented in order to elicit a more robust anti-HCV immune response. Recent nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both the cellular and humoral components of the immune system. This review focuses on recent advances in understanding the E1E2 heterodimeric structure to facilitate a rational design approach and the potential for development of a multivalent nanoparticle-based HCV E1E2 vaccine. Both aspects are considered important in the development of an effective HCV vaccine that can effectively address viral diversity and escape.