Publications by authors named "Neil P King"

50 Publications

Adjuvanting a subunit COVID-19 vaccine to induce protective immunity.

Nature 2021 Apr 19. Epub 2021 Apr 19.

Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford University, Stanford, CA, USA.

The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike receptor binding domain displayed on a protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing oil-in-water emulsion; AS37, a TLR-7 agonist adsorbed to Alum; CpG1018-Alum, a TLR-9 agonist formulated in Alum; and Alum. RBD-NP immunization with AS03, CpG1018-Alum, AS37 or Alum induced substantial nAb and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. Live-virus nAb response was maintained up to 180 days post-vaccination with RBD/AS03, and correlated with protection. RBD-NP immunization cross-neutralized the B.1.1.7 variant efficiently but showed a reduced response against the B.1.351 variant. While RBD-NP/AS03 demonstrated a 4.5-fold reduction in neutralization of B.1.351, the RBD-NP/AS37 group showed a 16-fold reduction, suggesting differences in the breadth of the nAb response induced by these adjuvants. Furthermore, RBD-NP/AS03 was as immunogenic as a prefusion stabilized Spike immunogen (Hexapro) adjuvanted with AS03. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2, and have paved the way for the clinical evaluation of this vaccine in Phase I/II clinical trials (NCT04742738 and NCT04750343).
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http://dx.doi.org/10.1038/s41586-021-03530-2DOI Listing
April 2021

Designed proteins assemble antibodies into modular nanocages.

Science 2021 04;372(6537)

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

Multivalent display of receptor-engaging antibodies or ligands can enhance their activity. Instead of achieving multivalency by attachment to preexisting scaffolds, here we unite form and function by the computational design of nanocages in which one structural component is an antibody or Fc-ligand fusion and the second is a designed antibody-binding homo-oligomer that drives nanocage assembly. Structures of eight nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage, respectively, closely match the corresponding computational models. Antibody nanocages targeting cell surface receptors enhance signaling compared with free antibodies or Fc-fusions in death receptor 5 (DR5)-mediated apoptosis, angiopoietin-1 receptor (Tie2)-mediated angiogenesis, CD40 activation, and T cell proliferation. Nanocage assembly also increases severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus neutralization by α-SARS-CoV-2 monoclonal antibodies and Fc-angiotensin-converting enzyme 2 (ACE2) fusion proteins.
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http://dx.doi.org/10.1126/science.abd9994DOI Listing
April 2021

Structure-based design of novel polyhedral protein nanomaterials.

Curr Opin Microbiol 2021 Mar 27;61:51-57. Epub 2021 Mar 27.

Department of Biochemistry and Institute for Protein Design, University of Washington, Seattle, WA, USA. Electronic address:

Organizing matter at the atomic scale is a central goal of nanotechnology. Bottom-up approaches, in which molecular building blocks are programmed to assemble via supramolecular interactions, are a proven and versatile route to new and useful nanomaterials. Although a wide variety of molecules have been used as building blocks, proteins have several intrinsic features that present unique opportunities for designing nanomaterials with sophisticated functions. There has been tremendous recent progress in designing proteins to fold and assemble to highly ordered structures. Here we review the leading approaches to the design of closed polyhedral protein assemblies, highlight the importance of considering the assembly process itself, and discuss various applications and future directions for the field. We emphasize throughout the exciting opportunities presented by recent advances as well as challenges that remain.
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http://dx.doi.org/10.1016/j.mib.2021.03.003DOI Listing
March 2021

Quadrivalent influenza nanoparticle vaccines induce broad protection.

Nature 2021 Mar 24. Epub 2021 Mar 24.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

Influenza vaccines that confer broad and durable protection against diverse viral strains would have a major effect on global health, as they would lessen the need for annual vaccine reformulation and immunization. Here we show that computationally designed, two-component nanoparticle immunogens induce potently neutralizing and broadly protective antibody responses against a wide variety of influenza viruses. The nanoparticle immunogens contain 20 haemagglutinin glycoprotein trimers in an ordered array, and their assembly in vitro enables the precisely controlled co-display of multiple distinct haemagglutinin proteins in defined ratios. Nanoparticle immunogens that co-display the four haemagglutinins of licensed quadrivalent influenza vaccines elicited antibody responses in several animal models against vaccine-matched strains that were equivalent to or better than commercial quadrivalent influenza vaccines, and simultaneously induced broadly protective antibody responses to heterologous viruses by targeting the subdominant yet conserved haemagglutinin stem. The combination of potent receptor-blocking and cross-reactive stem-directed antibodies induced by the nanoparticle immunogens makes them attractive candidates for a supraseasonal influenza vaccine candidate with the potential to replace conventional seasonal vaccines.
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http://dx.doi.org/10.1038/s41586-021-03365-xDOI Listing
March 2021

Engineered SARS-CoV-2 receptor binding domain improves immunogenicity in mice and elicits protective immunity in hamsters.

bioRxiv 2021 Mar 4. Epub 2021 Mar 4.

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing costs. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.
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http://dx.doi.org/10.1101/2021.03.03.433558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941618PMC
March 2021

Multimeric antibodies from antigen-specific human IgM+ memory B cells restrict Plasmodium parasites.

J Exp Med 2021 Apr;218(4)

Department of Immunology, University of Washington School of Medicine, Seattle, WA.

Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum-specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines.
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http://dx.doi.org/10.1084/jem.20200942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938364PMC
April 2021

Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates.

bioRxiv 2021 Feb 11. Epub 2021 Feb 11.

The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor-binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.
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http://dx.doi.org/10.1101/2021.02.10.430696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885918PMC
February 2021

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection.

Cell 2021 03 26;184(5):1188-1200.e19. Epub 2021 Jan 26.

Department of Medical Microbiology, Amsterdam UMC, University of Amsterdam, Amsterdam Infection & Immunity Institute, 1105 AZ Amsterdam, the Netherlands. Electronic address:

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication in the upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.
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http://dx.doi.org/10.1016/j.cell.2021.01.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7834972PMC
March 2021

Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations.

NPJ Vaccines 2020 Aug 5;5(1):72. Epub 2020 Aug 5.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.
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http://dx.doi.org/10.1038/s41541-020-00223-1DOI Listing
August 2020

Complete and cooperative in vitro assembly of computationally designed self-assembling protein nanomaterials.

Nat Commun 2021 02 9;12(1):883. Epub 2021 Feb 9.

Department of Biochemistry, University of Washington, Seattle, WA, USA.

Recent advances in computational methods have enabled the predictive design of self-assembling protein nanomaterials with atomic-level accuracy. These design strategies focus exclusively on a single target structure, without consideration of the mechanism or dynamics of assembly. However, understanding the assembly process, and in particular its robustness to perturbation, will be critical for translating this class of materials into useful technologies. Here we investigate the assembly of two computationally designed, 120-subunit icosahedral complexes in detail using several complementary biochemical methods. We found that assembly of each material from its two constituent protein building blocks was highly cooperative and yielded exclusively complete, 120-subunit complexes except in one non-stoichiometric regime for one of the materials. Our results suggest that in vitro assembly provides a robust and controllable route for the manufacture of designed protein nanomaterials and confirm that cooperative assembly can be an intrinsic, rather than evolved, feature of hierarchically structured protein complexes.
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http://dx.doi.org/10.1038/s41467-021-21251-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873210PMC
February 2021

Immunofocusing and enhancing autologous Tier-2 HIV-1 neutralization by displaying Env trimers on two-component protein nanoparticles.

NPJ Vaccines 2021 Feb 9;6(1):24. Epub 2021 Feb 9.

Department of Medical Microbiology, Amsterdam UMC, University of Amsterdam, Amsterdam Infection & Immunity Institute, Amsterdam, The Netherlands.

The HIV-1 envelope glycoprotein trimer is poorly immunogenic because it is covered by a dense glycan shield. As a result, recombinant Env glycoproteins generally elicit inadequate antibody levels that neutralize clinically relevant, neutralization-resistant (Tier-2) HIV-1 strains. Multivalent antigen presentation on nanoparticles is an established strategy to increase vaccine-driven immune responses. However, due to nanoparticle instability in vivo, the display of non-native Env structures, and the inaccessibility of many neutralizing antibody (NAb) epitopes, the effects of nanoparticle display are generally modest for Env trimers. Here, we generate two-component self-assembling protein nanoparticles presenting twenty SOSIP trimers of the clade C Tier-2 genotype 16055. We show in a rabbit immunization study that these nanoparticles induce 60-fold higher autologous Tier-2 NAb titers than the corresponding SOSIP trimers. Epitope mapping studies reveal that the presentation of 16055 SOSIP trimers on these nanoparticle focuses antibody responses to an immunodominant apical epitope. Thus, these nanoparticles are a promising platform to improve the immunogenicity of Env trimers with apex-proximate NAb epitopes.
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http://dx.doi.org/10.1038/s41541-021-00285-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873233PMC
February 2021

Dynamics of Neutralizing Antibody Titers in the Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

J Infect Dis 2021 02;223(2):197-205

Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Most individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop neutralizing antibodies that target the viral spike protein. In this study, we quantified how levels of these antibodies change in the months after SARS-CoV-2 infection by examining longitudinal samples collected approximately 30-152 days after symptom onset from a prospective cohort of 32 recovered individuals with asymptomatic, mild, or moderate-severe disease. Neutralizing antibody titers declined an average of about 4-fold from 1 to 4 months after symptom onset. This decline in neutralizing antibody titers was accompanied by a decline in total antibodies capable of binding the viral spike protein or its receptor-binding domain. Importantly, our data are consistent with the expected early immune response to viral infection, where an initial peak in antibody levels is followed by a decline to a lower plateau. Additional studies of long-lived B cells and antibody titers over longer time frames are necessary to determine the durability of immunity to SARS-CoV-2.
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http://dx.doi.org/10.1093/infdis/jiaa618DOI Listing
February 2021

Aldehyde Oxidase Contributes to All--Retinoic Acid Biosynthesis in Human Liver.

Drug Metab Dispos 2021 Mar 18;49(3):202-211. Epub 2020 Dec 18.

Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, Washington (G.Z., C.J.S., H.X., N.I.); Department of Chemistry, Washington State University, Pullman, Washington (E.M.P., J.P.J.); and Department of Biochemistry and Institute for Protein Design, University of Washington, Seattle, Washington (K.-L.H., N.P.K.)

All--retinoic acid (RA) is a critical endogenous signaling molecule. RA is predominantly synthesized from retinaldehyde by aldehyde dehydrogenase 1A1 (ALDH1A1), but aldehyde oxidase (AOX) may also contribute to RA biosynthesis. The goal of this study was to test the hypothesis that AOX contributes significantly to RA formation in human liver. Human recombinant AOX formed RA from retinaldehyde (K ∼1.5 ± 0.4 µM; k ∼3.6 ± 2.0 minute). In human liver S9 fractions (HLS9), RA formation was observed in the absence of NAD, suggesting AOX contribution to RA formation. In the presence of NAD, Eadie-Hofstee plots of RA formation in HLS9 indicated that two enzymes contributed to RA formation. The two enzymes were identified as AOX and ALDH1A1 based on inhibition of RA formation by AOX inhibitor hydralazine (20%-50% inhibition) and ALDH1A1 inhibitor WIN18,446 (50%-80%inhibition). The expression of AOX in HLS9 was 9.4-24 pmol mg S9 protein, whereas ALDH1A1 expression was 156-285 pmol mg S9 protein measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of signature peptides. The formation velocity of RA in the presence of NAD correlated significantly with the expression of ALDH1A1 and AOX protein. Taken together, the data show that both AOX and ALDH1A1 contribute to RA biosynthesis in the human liver, with ALDH1A1 being the high-affinity, low-capacity enzyme and AOX being the low-affinity, high-capacity enzyme. The results suggest that in the case of ALDH1A dysfunction or excess vitamin A, AOX may play an important role in regulating hepatic vitamin A homeostasis and that inhibition of AOX may alter RA biosynthesis and signaling. SIGNIFICANCE STATEMENT: This study provides direct evidence to show that human AOX converts retinaldehyde to RA and contributes to hepatic RA biosynthesis. The finding that AOX may be responsible for 20%-50% of overall hepatic RA formation suggests that alterations in AOX activity via drug-drug interactions, genetic polymorphisms, or disease states may impact hepatic RA concentrations and signaling and alter vitamin A homeostasis.
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http://dx.doi.org/10.1124/dmd.120.000296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885020PMC
March 2021

Designed proteins assemble antibodies into modular nanocages.

bioRxiv 2020 Dec 1. Epub 2020 Dec 1.

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

Antibodies are widely used in biology and medicine, and there has been considerable interest in multivalent antibody formats to increase binding avidity and enhance signaling pathway agonism. However, there are currently no general approaches for forming precisely oriented antibody assemblies with controlled valency. We describe the computational design of two-component nanocages that overcome this limitation by uniting form and function. One structural component is any antibody or Fc fusion and the second is a designed Fc-binding homo-oligomer that drives nanocage assembly. Structures of 8 antibody nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage match the corresponding computational models. Antibody nanocages targeting cell-surface receptors enhance signaling compared to free antibodies or Fc-fusions in DR5-mediated apoptosis, Tie2-mediated angiogenesis, CD40 activation, and T cell proliferation; nanocage assembly also increases SARS-CoV-2 pseudovirus neutralization by α-SARS-CoV-2 monoclonal antibodies and Fc-ACE2 fusion proteins. We anticipate that the ability to assemble arbitrary antibodies without need for covalent modification into highly ordered assemblies with different geometries and valencies will have broad impact in biology and medicine.
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http://dx.doi.org/10.1101/2020.12.01.406611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724662PMC
December 2020

Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19.

Cell 2021 01 23;184(1):169-183.e17. Epub 2020 Nov 23.

Department of Immunology, University of Washington School of Medicine, Seattle, WA 98109, USA. Electronic address:

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is causing a global pandemic, and cases continue to rise. Most infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that could contribute to immunity. We performed a longitudinal assessment of individuals recovered from mild COVID-19 to determine whether they develop and sustain multifaceted SARS-CoV-2-specific immunological memory. Recovered individuals developed SARS-CoV-2-specific immunoglobulin (IgG) antibodies, neutralizing plasma, and memory B and memory T cells that persisted for at least 3 months. Our data further reveal that SARS-CoV-2-specific IgG memory B cells increased over time. Additionally, SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral function: memory T cells secreted cytokines and expanded upon antigen re-encounter, whereas memory B cells expressed receptors capable of neutralizing virus when expressed as monoclonal antibodies. Therefore, mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks of antiviral immunity.
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http://dx.doi.org/10.1016/j.cell.2020.11.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682481PMC
January 2021

Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2.

Cell 2020 11 31;183(5):1367-1382.e17. Epub 2020 Oct 31.

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA; Institute for Protein Design, University of Washington, Seattle, WA 98195, USA. Electronic address:

A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike despite a 5-fold lower dose. Antibodies elicited by the RBD nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufacturing efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic.
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http://dx.doi.org/10.1016/j.cell.2020.10.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604136PMC
November 2020

Dynamics of neutralizing antibody titers in the months after SARS-CoV-2 infection.

J Infect Dis 2020 Sep 30. Epub 2020 Sep 30.

Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Most individuals infected with SARS-CoV-2 develop neutralizing antibodies that target the viral spike protein. Here we quantify how levels of these antibodies change in the months following SARS-CoV-2 infection by examining longitudinal samples collected between ~30 and 152 days post symptom onset from a prospective cohort of 32 recovered individuals with asymptomatic, mild, or moderate-severe disease. Neutralizing antibody titers declined an average of about four-fold from one to four months post symptom onset. This decline in neutralizing antibody titers was accompanied by a decline in total antibodies capable of binding the viral spike or its receptor-binding domain. Importantly, our data are consistent with the expected early immune response to viral infection, where an initial peak in antibody levels is followed by a decline to a lower plateau. Additional studies of long-lived B-cells and antibody titers over longer time frames are necessary to determine the durability of immunity to SARS-CoV-2.
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http://dx.doi.org/10.1093/infdis/jiaa618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7543487PMC
September 2020

A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver.

Immunity 2020 10 17;53(4):733-744.e8. Epub 2020 Sep 17.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.
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http://dx.doi.org/10.1016/j.immuni.2020.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572793PMC
October 2020

Serological identification of SARS-CoV-2 infections among children visiting a hospital during the initial Seattle outbreak.

Nat Commun 2020 09 1;11(1):4378. Epub 2020 Sep 1.

Basic Sciences and Computational Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA.

Children are strikingly underrepresented in COVID-19 case counts. In the United States, children represent 22% of the population but only 1.7% of confirmed SARS-CoV-2 cases as of April 2, 2020. One possibility is that symptom-based viral testing is less likely to identify infected children, since they often experience milder disease than adults. Here, to better assess the frequency of pediatric SARS-CoV-2 infection, we serologically screen 1,775 residual samples from Seattle Children's Hospital collected from 1,076 children seeking medical care during March and April of 2020. Only one child was seropositive in March, but seven were seropositive in April for a period seroprevalence of ≈1%. Most seropositive children (6/8) were not suspected of having had COVID-19. The sera of seropositive children have neutralizing activity, including one that neutralized at a dilution > 1:18,000. Therefore, an increasing number of children seeking medical care were infected by SARS-CoV-2 during the early Seattle outbreak despite few positive viral tests.
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http://dx.doi.org/10.1038/s41467-020-18178-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463158PMC
September 2020

Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding.

Cell 2020 09 11;182(5):1295-1310.e20. Epub 2020 Aug 11.

Basic Sciences Division and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA; Howard Hughes Medical Institute, Seattle, WA 98109, USA. Electronic address:

The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor and is a major determinant of host range and a dominant target of neutralizing antibodies. Here, we experimentally measure how all amino acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD's surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity-enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open analysis pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations observed during viral surveillance.
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http://dx.doi.org/10.1016/j.cell.2020.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418704PMC
September 2020

Functional SARS-CoV-2-specific immune memory persists after mild COVID-19.

Res Sq 2020 Aug 13. Epub 2020 Aug 13.

Department of Immunology, University of Washington School of Medicine, Seattle, WA, USA.

The recently emerged SARS-CoV-2 virus is currently causing a global pandemic and cases continue to rise. The majority of infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that might contribute to herd immunity. Thus, we performed a longitudinal assessment of individuals recovered from mildly symptomatic COVID-19 to determine if they develop and sustain immunological memory against the virus. We found that recovered individuals developed SARS-CoV-2-specific IgG antibody and neutralizing plasma, as well as virus-specific memory B and T cells that not only persisted, but in some cases increased numerically over three months following symptom onset. Furthermore, the SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral immunity: memory T cells secreted IFN-γ and expanded upon antigen re-encounter, while memory B cells expressed receptors capable of neutralizing virus when expressed as antibodies. These findings demonstrate that mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks associated with antiviral protective immunity.
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http://dx.doi.org/10.21203/rs.3.rs-57112/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430600PMC
August 2020

Functional SARS-CoV-2-specific immune memory persists after mild COVID-19.

medRxiv 2020 Aug 15. Epub 2020 Aug 15.

The recently emerged SARS-CoV-2 virus is currently causing a global pandemic and cases continue to rise. The majority of infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that might contribute to herd immunity. Thus, we performed a longitudinal assessment of individuals recovered from mildly symptomatic COVID-19 to determine if they develop and sustain immunological memory against the virus. We found that recovered individuals developed SARS-CoV-2-specific IgG antibody and neutralizing plasma, as well as virus-specific memory B and T cells that not only persisted, but in some cases increased numerically over three months following symptom onset. Furthermore, the SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral immunity: memory T cells secreted IFN-γ and expanded upon antigen re-encounter, while memory B cells expressed receptors capable of neutralizing virus when expressed as antibodies. These findings demonstrate that mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks associated with antiviral protective immunity.
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http://dx.doi.org/10.1101/2020.08.11.20171843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430599PMC
August 2020

Elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for SARS-CoV-2.

bioRxiv 2020 Aug 12. Epub 2020 Aug 12.

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, we describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms for inducing potent neutralizing antibody responses and have launched cGMP manufacturing efforts to advance the lead RBD nanoparticle vaccine into the clinic.
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http://dx.doi.org/10.1101/2020.08.11.247395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430571PMC
August 2020

Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations.

NPJ Vaccines 2020 5;5:72. Epub 2020 Aug 5.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 USA.

Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.
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http://dx.doi.org/10.1038/s41541-020-00223-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406516PMC
August 2020

Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens.

PLoS Pathog 2020 08 11;16(8):e1008665. Epub 2020 Aug 11.

Department of Integrative, Structural and Computational Biology, Scripps Research, La Jolla, California, United States of America.

Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and geometries enables combination with antigens of choice to test novel multimerization concepts in immunization strategies where the goal is to improve the induction and maturation of neutralizing antibody lineages. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to an underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. When the immunogenicity of ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle or as soluble proteins were compared in rabbits, the two immunogens elicited similar serum antibody binding titers against the trimer component. Neutralizing antibody titers were slightly elevated in the animals given the nanoparticle immunogen and were initially more focused to the trimer apex. Altogether, our findings indicate that tetrahedral nanoparticles can be successfully applied for presentation of HIV Env trimer immunogens; however, the optimal implementation to different immunization strategies remains to be determined.
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http://dx.doi.org/10.1371/journal.ppat.1008665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418955PMC
August 2020

Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens.

Elife 2020 08 4;9. Epub 2020 Aug 4.

Department of Biochemistry, University of Washington, Seattle, United States.

Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. To enable a new generation of anti-viral vaccines, we designed self-assembling protein nanoparticles with geometries tailored to present the ectodomains of influenza, HIV, and RSV viral glycoprotein trimers. We first designed trimers tailored for antigen fusion, featuring N-terminal helices positioned to match the C termini of the viral glycoproteins. Trimers that experimentally adopted their designed configurations were incorporated as components of tetrahedral, octahedral, and icosahedral nanoparticles, which were characterized by cryo-electron microscopy and assessed for their ability to present viral glycoproteins. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles presented antigenically intact prefusion HIV-1 Env, influenza hemagglutinin, and RSV F trimers in the predicted geometries. This work demonstrates that antigen-displaying protein nanoparticles can be designed from scratch, and provides a systematic way to investigate the influence of antigen presentation geometry on the immune response to vaccination.
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http://dx.doi.org/10.7554/eLife.57659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402677PMC
August 2020

Seroprevalence of SARS-CoV-2 among children visiting a hospital during the initial Seattle outbreak.

medRxiv 2020 May 28. Epub 2020 May 28.

Basic Sciences and Computational Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Children are strikingly underrepresented in COVID-19 case counts. In the United States, children represent 22% of the population but only 1.7% of confirmed SARS-CoV-2 cases. One possibility is that symptom-based viral testing is less likely to identify infected children, since they often experience milder disease than adults. To better assess the frequency of pediatric SARS-CoV-2 infection, we serologically screened 1,775 residual samples from Seattle Children's Hospital collected from 1,076 children seeking medical care during March and April of 2020. Only one child was seropositive in March, but nine were seropositive in April for a period seroprevalence of >1%. Most seropositive children (8/10) were not suspected of having had COVID-19. The sera of most seropositive children had neutralizing activity, including one that neutralized at a dilution >1:18,000. Therefore, among children seeking medical care, the frequency of SARS-CoV-2 infection increased markedly during the early Seattle outbreak despite few positive viral tests.
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http://dx.doi.org/10.1101/2020.05.26.20114124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273251PMC
May 2020

Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays.

Viruses 2020 05 6;12(5). Epub 2020 May 6.

Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

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 vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed 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 made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could 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.
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http://dx.doi.org/10.3390/v12050513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291041PMC
May 2020

Design and structure of two new protein cages illustrate successes and ongoing challenges in protein engineering.

Protein Sci 2020 04 26;29(4):919-929. Epub 2019 Dec 26.

UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, California.

In recent years, new protein engineering methods have produced more than a dozen symmetric, self-assembling protein cages whose structures have been validated to match their design models with near-atomic accuracy. However, many protein cage designs that are tested in the lab do not form the desired assembly, and improving the success rate of design has been a point of recent emphasis. Here we present two protein structures solved by X-ray crystallography of designed protein oligomers that form two-component cages with tetrahedral symmetry. To improve on the past tendency toward poorly soluble protein, we used a computational protocol that favors the formation of hydrogen-bonding networks over exclusively hydrophobic interactions to stabilize the designed protein-protein interfaces. Preliminary characterization showed highly soluble expression, and solution studies indicated successful cage formation by both designed proteins. For one of the designs, a crystal structure confirmed at high resolution that the intended tetrahedral cage was formed, though several flipped amino acid side chain rotamers resulted in an interface that deviates from the precise hydrogen-bonding pattern that was intended. A structure of the other designed cage showed that, under the conditions where crystals were obtained, a noncage structure was formed wherein a porous 3D protein network in space group I2 3 is generated by an off-target twofold homomeric interface. These results illustrate some of the ongoing challenges of developing computational methods for polar interface design, and add two potentially valuable new entries to the growing list of engineered protein materials for downstream applications.
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http://dx.doi.org/10.1002/pro.3802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096701PMC
April 2020

Enhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle.

Nat Commun 2019 09 19;10(1):4272. Epub 2019 Sep 19.

Amsterdam UMC, Department of Medical Microbiology, Amsterdam Infection & Immunity Institute, University of Amsterdam, Amsterdam, 1105AZ, The Netherlands.

The development of native-like HIV-1 envelope (Env) trimer antigens has enabled the induction of neutralizing antibody (NAb) responses against neutralization-resistant HIV-1 strains in animal models. However, NAb responses are relatively weak and narrow in specificity. Displaying antigens in a multivalent fashion on nanoparticles (NPs) is an established strategy to increase their immunogenicity. Here we present the design and characterization of two-component protein NPs displaying 20 stabilized SOSIP trimers from various HIV-1 strains. The two-component nature permits the incorporation of exclusively well-folded, native-like Env trimers into NPs that self-assemble in vitro with high efficiency. Immunization studies show that the NPs are particularly efficacious as priming immunogens, improve the quality of the Ab response over a conventional one-component nanoparticle system, and are most effective when SOSIP trimers with an apex-proximate neutralizing epitope are displayed. Their ability to enhance and shape the immunogenicity of SOSIP trimers make these NPs a promising immunogen platform.
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http://dx.doi.org/10.1038/s41467-019-12080-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753213PMC
September 2019