Neil P. King

University of Washington

Author Statistics

Papers

Citation

H-Index

i-10 index

Research Trends

Author Order

Document Type

Co-Authors

David Baker

University of Washington

David Veesler

University of Washington

Lauren Carter

University of Washington

Rashmi Ravichandran

University of Washington

Brooke Fiala

University of Washington

William Sheffler

University of Washington

Daniel Ellis

University of Washington

Marcos C. Miranda

University of Washington

Michael Murphy

University of Washington

Deleah Pettie

University of Washington

Cooperative Institutions

University of Washington

394

Seattle University

134

New York Academy of Sciences

128

Hudson Institute

127

John Wiley & Sons (Germany)

124

Moss Landing Marine Laboratories

123

National Institutes of Health

102

National Institute of Allergy and Infectious Diseases

Fred Hutch Cancer Center

Scripps Research Institute

Author Statistics

Papers

Citation

H-Index

i-10 index

Research Field

De novo design of tunable, pH-driven conformational changes

Science (2019)

Scott E. Boyken Mark A. Benhaim Florian Büsch Mengxuan Jia Matthew J. Bick

The ability of naturally occurring proteins to change conformation in response to environmental changes is critical to biological function. Although there have been advances in the de novo design of stable proteins with a single, deep free-energy minimum, the design of conformational switches remains challenging. We present a general strategy to design pH-responsive protein conformational changes by precisely preorganizing histidine residues in buried hydrogen-bond networks. We design homotrimers and heterodimers that are stable above pH 6.5 but undergo cooperative, large-scale conformational changes when the pH is lowered and electrostatic and steric repulsion builds up as the network histidine residues become protonated. The transition pH and cooperativity can be controlled through the number of histidine-containing networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt lipid membranes both in vitro and after being endocytosed in mammalian cells. Our results demonstrate that environmentally triggered conformational changes can now be programmed by de novo protein design.

Cooperativity

Conformational change

Protein design

10.1126/science.aav7897

Cite

Citations (137)

Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays

bioRxiv (Cold Spring Harbor Laboratory) (2020)

Katharine H. D. Crawford Rachel Eguia Adam S. Dingens Andrea N. Loes Keara D. Malone

Abstract SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral and VSV particles, but the reagents and protocols are not widely available. Here we detail how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

Biosafety

spike protein

Coronavirus

HEK 293 cells

10.1101/2020.04.20.051219

Cite

Citations (156)

Protein nanoparticle vaccines induce potent neutralizing antibody responses against MERS-CoV

bioRxiv (Cold Spring Harbor Laboratory) (2024)

Cara W. Chao Kaitlin R. Sprouse Marcos C. Miranda Nicholas Catanzaro Miranda L. Hubbard

Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that causes severe and often lethal respiratory illness in humans. The MERS-CoV spike (S) protein is the viral fusogen and the target of neutralizing antibodies, and has therefore been the focus of vaccine design efforts. Currently there are no licensed vaccines against MERS-CoV and only a few candidates have advanced to Phase I clinical trials. Here we developed MERS-CoV vaccines utilizing a computationally designed protein nanoparticle platform that has generated safe and immunogenic vaccines against various enveloped viruses, including a licensed vaccine for SARS-CoV-2. Two-component protein nanoparticles displaying MERS-CoV S-derived antigens induced robust neutralizing antibody responses and protected mice against challenge with mouse-adapted MERS-CoV. Electron microscopy polyclonal epitope mapping and serum competition assays revealed the specificities of the dominant antibody responses elicited by immunogens displaying the prefusion-stabilized S-2P trimer, receptor binding domain (RBD), or N-terminal domain (NTD). An RBD nanoparticle vaccine elicited antibodies targeting multiple non-overlapping epitopes in the RBD, whereas anti-NTD antibodies elicited by the S-2P– and NTD-based immunogens converged on a single antigenic site. Our findings demonstrate the potential of two-component nanoparticle vaccine candidates for MERS-CoV and suggest that this platform technology could be broadly applicable to betacoronavirus vaccine development.

Polyclonal antibodies

Epitope mapping

Betacoronavirus

Coronavirus

Conformational epitope

10.1101/2024.03.13.584735

Cite

Citations (2)

Distinct sensitivities to SARS-CoV-2 variants in vaccinated humans and mice

bioRxiv (Cold Spring Harbor Laboratory) (2022)

Alexandra C. Walls Laura A. VanBlargan Kaichun Wu Angela Choi Mary Jane Navarro

Abstract The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has led to the development of a large number of vaccines, several of which are now approved for use in humans. Understanding vaccine-elicited antibody responses against emerging SARS-CoV-2 variants of concern (VOC) in real time is key to inform public health policies. Serum neutralizing antibody titers are the current best correlate of protection from SARS-CoV-2 challenge in non-human primates and a key metric to understand immune evasion of VOC. We report that vaccinated BALB/c mice do not recapitulate faithfully the breadth and potency of neutralizing antibody responses against VOC, as compared to non-human primates or humans, suggesting caution should be exercised when interpreting data for this animal model.

Antibody response

2019-20 coronavirus outbreak

Coronavirus

Antibody titer

10.1101/2022.02.07.479468

Cite

Citations (1)

Vaccination with a structure-based stabilized version of malarial antigen Pfs48/45 elicits ultra-potent transmission-blocking antibody responses

Immunity (2022)

Brandon McLeod Moustafa T. Mabrouk Kazutoyo Miura Rashmi Ravichandran Sally Kephart

Malaria transmission-blocking vaccines (TBVs) aim to elicit human antibodies that inhibit sporogonic development of Plasmodium falciparum in mosquitoes, thereby preventing onward transmission. Pfs48/45 is a leading clinical TBV candidate antigen and is recognized by the most potent transmission-blocking monoclonal antibody (mAb) yet described; still, clinical development of Pfs48/45 antigens has been hindered, largely by its poor biochemical characteristics. Here, we used structure-based computational approaches to design Pfs48/45 antigens stabilized in the conformation recognized by the most potently inhibitory mAb, achieving >25°C higher thermostability compared with the wild-type protein. Antibodies elicited in mice immunized with these engineered antigens displayed on liposome-based or protein nanoparticle-based vaccine platforms exhibited 1–2 orders of magnitude superior transmission-reducing activity, compared with immunogens bearing the wild-type antigen, driven by improved antibody quality. Our data provide the founding principles for using molecular stabilization solely from antibody structure-function information to drive improved immune responses against a parasitic vaccine target.

Antibody response

Blocking (statistics)

Blocking antibody

10.1016/j.immuni.2022.07.015

Cite

Citations (40)

Principles for designing ordered protein assemblies

Trends in Cell Biology (2012)

Yen‐Ting Lai Neil P. King Todd O. Yeates

10.1016/j.tcb.2012.08.004

Cite

Citations (130)

Structure, receptor recognition, and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein

Cell (2022)

M. Alejandra Tortorici Alexandra C. Walls Anshu Joshi Young‐Jun Park Rachel Eguia

Coronavirus

Antigenicity

Polyclonal antibodies

10.1016/j.cell.2022.05.019

Cite

Citations (57)

Computationally Designed Self-assembling tetrahedron protein, T310

M.R. Sawaya Neil P. King William Sheffler David Baker Todd O. Yeates

Tetrahedron

10.2210/pdb4egg/pdb

Cite

Citations (0)

Antigen- and scaffold-specific antibody responses to protein nanoparticle immunogens

Cell Reports Medicine (2022)

John C. Kraft Minh N. Pham Laila Shehata Mitch Brinkkemper Seyhan Boyoglu-Barnum

Protein nanoparticle scaffolds are increasingly used in next-generation vaccine designs, and several have established records of clinical safety and efficacy. Yet the rules for how immune responses specific to nanoparticle scaffolds affect the immunogenicity of displayed antigens have not been established. Here we define relationships between anti-scaffold and antigen-specific antibody responses elicited by protein nanoparticle immunogens. We report that dampening anti-scaffold responses by physical masking does not enhance antigen-specific antibody responses. In a series of immunogens that all use the same nanoparticle scaffold but display four different antigens, only HIV-1 envelope glycoprotein (Env) is subdominant to the scaffold. However, we also demonstrate that scaffold-specific antibody responses can competitively inhibit antigen-specific responses when the scaffold is provided in excess. Overall, our results suggest that anti-scaffold antibody responses are unlikely to suppress antigen-specific antibody responses for protein nanoparticle immunogens in which the antigen is immunodominant over the scaffold.

10.1016/j.xcrm.2022.100780

Cite

Citations (31)

Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM

bioRxiv (Cold Spring Harbor Laboratory) (2021)

Aleksandar Antanasijevic Leigh M. Sewall Christopher A. Cottrell Diane G. Carnathan Luis Jiménez

In Brief Herein, we evaluated the immunogenicity of several BG505 SOSIP-based HIV Env immunogens in the rhesus macaque animal model using a combination of serology and biophysical approaches. We applied electron cryo-microscopy for high-resolution mapping of elicited polyclonal antibody responses, which provided detailed insights into the binding modes of the most common classes of antibodies elicited by BG505 SOSIP immunogens as well as the critical differences in immunogenicity that can occur as a consequence of engineered stabilizing mutations and partial glycan occupancy at different sites. Summary Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic. Graphical abstract

Polyclonal antibodies

Epitope mapping

HIV vaccine

10.1101/2021.01.28.428677

Cite

Citations (3)