King Lab | Publications

2024

• Hierarchical design of pseudosymmetric protein nanocages
  Matdes Nanoparticles Pseudosymmetry Lab-led
  Dowling QM, Park YJ, Fries CN, Gerstenmaier NC, Ols S, Yang EC, Wargacki AJ, Dosey A, Hsia Y, Ravichandran R, Walkey CD, Burrell AL, Veesler D, Baker D, King NP. | doi:10.1038/s41586-024-08360-6 PDF Abstract
  Discrete protein assemblies ranging from hundreds of kilodaltons to hundreds of megadaltons in size are a ubiquitous feature of biological systems and perform highly specialized functions. Despite remarkable recent progress in accurately designing new self-assembling proteins, the size and complexity of these assemblies has been limited by a reliance on strict symmetry. Here, inspired by the pseudosymmetry observed in bacterial microcompartments and viral capsids, we developed a hierarchical computational method for designing large pseudosymmetric self-assembling protein nanomaterials. We computationally designed pseudosymmetric heterooligomeric components and used them to create discrete, cage-like protein assemblies with icosahedral symmetry containing 240, 540 and 960 subunits. At 49, 71 and 96 nm diameter, these nanocages are the largest bounded computationally designed protein assemblies generated to date. More broadly, by moving beyond strict symmetry, our work substantially broadens the variety of self-assembling protein architectures that are accessible through design.
• Protein nanoparticle vaccines induce potent neutralizing antibody responses against MERS-CoV
  Vaccines Nanoparticles Lab-led
  Chao CW, Sprouse KR, Miranda MC, Catanzaro NJ, Hubbard ML, Addetia A, Stewart C, Brown JT, Dosey A, Valdez A, Ravichandran R, Hendricks GG, Ahlrichs M, Dobbins C, Hand A, McGowan J, Simmons B, Treichel C, Willoughby I, Walls AC, McGuire AT, Leaf EM, Baric RS, Schäfer A, Veesler D, King NP.
  (2024) Cell Rep. 43(12):115036 | doi:10.1016/j.celrep.2024.115036 PDF Abstract
  Middle East respiratory syndrome coronavirus (MERS-CoV) is a betacoronavirus that causes severe respiratory illness in humans. There are no licensed vaccines against MERS-CoV and only a few candidates in phase I clinical trials. Here, we develop 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 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two-component nanoparticles displaying spike (S)-derived antigens induce neutralizing responses and protect mice against challenge with mouse-adapted MERS-CoV. Epitope mapping reveals the dominant responses elicited by immunogens displaying the prefusion-stabilized S-2P trimer, receptor binding domain (RBD), or N-terminal domain (NTD). An RBD nanoparticle elicits antibodies targeting multiple non-overlapping epitopes in the RBD. 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.
• Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance
  Dadonaite B, Ahn JJ, Ort JT, Yu J, Furey C, Dosey A, Hannon WW, Vincent Baker AL, Webby RJ, King NP, Liu Y, Hensley SE, Peacock TP, Moncla LH, Bloom JD.
  (2024) PLoS Biol. 22(11):e3002916 | doi:10.1371/journal.pbio.3002916 Abstract
  H5 influenza is considered a potential pandemic threat. Recently, H5 viruses belonging to clade 2.3.4.4b have caused large outbreaks in avian and multiple nonhuman mammalian species. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic potential in humans, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. However, prior experimental work has only measured how these phenotypes are affected by a handful of the >10,000 different possible amino-acid mutations to HA. Here, we use pseudovirus deep mutational scanning to measure how all mutations to a 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind α2-6-linked sialic acids and show that some viruses already carry mutations that stabilize HA. We also measure how all HA mutations affect neutralization by sera from mice and ferrets vaccinated against or infected with 2.3.4.4b H5 viruses. These antigenic maps enable rapid assessment of when new viral strains have acquired mutations that may create mismatches with candidate vaccine virus, and we show that a mutation present in some recent H5 HAs causes a large antigenic change. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive measurements of the phenotypic effects of mutations that can inform real-time interpretation of viral variation observed during surveillance of H5 influenza.
• Potent neutralization of SARS-CoV-2 variants by RBD nanoparticle and prefusion-stabilized spike immunogens
  Vaccines Nanoparticles Lab-led
  Miranda MC, Kepl E, Navarro MJ, Chen C, Johnson M, Sprouse KR, Stewart C, Palser A, Valdez A, Pettie D, Sydeman C, Ogohara C, Kraft JC, Pham M, Murphy M, Wrenn S, Fiala B, Ravichandran R, Ellis D, Carter L, Corti D, Kellam P, Lee K, Walls AC, Veesler D, King NP.
  (2024) NPJ Vaccines. 9(1):184 | doi:10.1038/s41541-024-00982-1 Abstract
  We previously described a two-component protein nanoparticle vaccine platform that displays 60 copies of the SARS-CoV-2 spike protein RBD (RBD-NP). The vaccine, when adjuvanted with AS03, was shown to elicit robust neutralizing antibody and CD4 T cell responses in Phase I/II clinical trials, met its primary co-endpoints in a Phase III trial, and has been licensed by multiple regulatory authorities under the brand name SKYCovione. Here we characterize the biophysical properties, stability, antigenicity, and immunogenicity of RBD-NP immunogens incorporating mutations from the B.1.351 (β) and P.1 (γ) variants of concern (VOCs) that emerged in 2020. We also show that the RBD-NP platform can be adapted to the Omicron strains BA.5 and XBB.1.5. We compare β and γ variant and E484K point mutant nanoparticle immunogens to the nanoparticle displaying the Wu-1 RBD, as well as to soluble prefusion-stabilized (HexaPro) spike trimers harboring VOC-derived mutations. We find the properties of immunogens based on different SARS-CoV-2 variants can differ substantially, which could affect the viability of variant vaccine development. Introducing stabilizing mutations in the linoleic acid binding site of the RBD-NPs resulted in increased physical stability compared to versions lacking the stabilizing mutations without deleteriously affecting immunogenicity. The RBD-NP immunogens and HexaPro trimers, as well as combinations of VOC-based immunogens, elicited comparable levels of neutralizing antibodies against distinct VOCs. Our results demonstrate that RBD-NP-based vaccines can elicit neutralizing antibody responses against SARS-CoV-2 variants and can be rapidly designed and stabilized, demonstrating the potential of two-component RBD-NPs as a platform for the development of broadly protective coronavirus vaccines.
• Mosaic and mixed HIV-1 glycoprotein nanoparticles elicit antibody responses to broadly neutralizing epitopes
  Brinkkemper M, Kerster G, Brouwer PJM, Tran AS, Torres JL, Ettema RA, Nijhuis H, Allen JD, Zhu W, Gao H, Lee WH, Bijl TPL, Snitselaar JL, Burger JA, Bontjer I, Olijhoek W, Ravichandran R, van Breemen MJ, Del Moral-Sánchez I, Derking R, Sliepen K, Ozorowski G, Crispin M, Montefiori DC, Claireaux M, Ward AB, van Gils MJ, King NP, Sanders RW.
  (2024) PLoS Pathog. 20(10):e1012558 | doi:10.1371/journal.ppat.1012558 Abstract
  An effective human immunodeficiency virus 1 (HIV-1) vaccine will most likely have to elicit broadly neutralizing antibodies (bNAbs) to overcome the sequence diversity of the envelope glycoprotein (Env). So far, stabilized versions of Env, such as SOSIP trimers, have been able to induce neutralizing antibody (NAb) responses, but those responses are mainly strain-specific. Here we attempted to broaden NAb responses by using a multivalent vaccine and applying a number of design improvements. First, we used highly stabilized SOSIP.v9 trimers. Second, we removed any holes in the glycan shields and optimized glycan occupancy to avoid strain-specific glycan hole responses. Third, we selected five sequences from the same clade (B), as we observed previously that combining Env trimers from clade A, B and C did not improve cross-reactive responses, as they might have been too diverse. Fourth, to improve antibody (Ab) responses, the Env trimers were displayed on two-component I53-50 nanoparticles (NPs). Fifth, to favor activation of cross-reactive B cells, the five Env trimers were co-displayed on mosaic NPs. Sixth, we immunized rabbits four times with long intervals between vaccinations. These efforts led to the induction of cross-reactive B cells and cross-reactive binding Ab responses, but we only sporadically detected cross-neutralizing responses. We conclude that stabilized HIV-1 Env trimers that are not modified specifically for priming naive B cells are unable to elicit strong bNAb responses, and infer that sequential immunization regimens, most likely starting with specific germline-targeting immunogens, will be necessary to overcome Env's defenses against the induction of NAbs. The antigens described here could be excellent boosting immunogens in a sequential immunization regimen, as responses to bNAb epitopes were induced.
• Saponin nanoparticle adjuvants incorporating Toll-like receptor agonists drive distinct immune signatures and potent vaccine responses
  Ou BS, Baillet J, Filsinger Interrante MV, Adamska JZ, Zhou X, Saouaf OM, Yan J, Klich JH, Jons CK, Meany EL, Valdez AS, Carter L, Pulendran B, King NP, Appel EA.
  (2024) Sci Adv. 10(32):eadn7187 | doi:10.1126/sciadv.adn7187 Abstract
  Over the past few decades, the development of potent and safe immune-activating adjuvant technologies has become the heart of intensive research in the constant fight against highly mutative and immune evasive viruses such as influenza, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and human immunodeficiency virus (HIV). Herein, we developed a highly modular saponin-based nanoparticle platform incorporating Toll-like receptor agonists (TLRas) including TLR1/2a, TLR4a, and TLR7/8a adjuvants and their mixtures. These various TLRa-saponin nanoparticle adjuvant constructs induce unique acute cytokine and immune-signaling profiles, leading to specific T helper responses that could be of interest depending on the target disease for prevention. In a murine vaccine study, the adjuvants greatly improved the potency, durability, breadth, and neutralization of both COVID-19 and HIV vaccine candidates, suggesting the potential broad application of these adjuvant constructs to a range of different antigens. Overall, this work demonstrates a modular TLRa-SNP adjuvant platform that could improve the design of vaccines and affect modern vaccine development.
• Deep mutational scanning reveals functional constraints and antibody-escape potential of Lassa virus glycoprotein complex
  Carr CR, Crawford KHD, Murphy M, Galloway JG, Haddox HK, Matsen FA, Andersen KG, King NP, Bloom JD.
  (2024) Immunity. 57(9):2061-2076.e11 | doi:10.1016/j.immuni.2024.06.013 Abstract
  Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of the Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we used pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affected cell entry and antibody neutralization. Our experiments defined functional constraints throughout GPC. We quantified how GPC mutations affected neutralization with a panel of monoclonal antibodies. All antibodies tested were escaped by mutations that existed among natural Lassa virus lineages. Overall, our work describes a biosafety-level-2 method to elucidate the mutational space accessible to GPC and shows how prospective characterization of antigenic variation could aid the design of therapeutics and vaccines.
• A broadly generalizable stabilization strategy for sarbecovirus fusion machinery vaccines
  Lee J, Stewart C, Schäfer A, Leaf EM, Park YJ, Asarnow D, Powers JM, Treichel C, Sprouse KR, Corti D, Baric R, King NP, Veesler D.
  (2024) Nat Commun. 15(1):5496 | doi:10.1038/s41467-024-49656-5 Abstract
  Evolution of SARS-CoV-2 alters the antigenicity of the immunodominant spike (S) receptor-binding domain and N-terminal domain, undermining the efficacy of vaccines and antibody therapies. To overcome this challenge, we set out to develop a vaccine focusing antibody responses on the highly conserved but metastable S subunit, which folds as a spring-loaded fusion machinery. We describe a strategy for prefusion-stabilization and high yield recombinant production of SARS-CoV-2 S trimers with native structure and antigenicity. We demonstrate that our design strategy is broadly generalizable to sarbecoviruses, as exemplified with the SARS-CoV-1 (clade 1a) and PRD-0038 (clade 3) S subunits. Immunization of mice with a prefusion-stabilized SARS-CoV-2 S trimer elicits broadly reactive sarbecovirus antibodies and neutralizing antibody titers of comparable magnitude against Wuhan-Hu-1 and the immune evasive XBB.1.5 variant. Vaccinated mice were protected from weight loss and disease upon challenge with XBB.1.5, providing proof-of-principle for fusion machinery sarbecovirus vaccines.
• A gH/gL-encoding replicon vaccine elicits neutralizing antibodies that protect humanized mice against EBV challenge
  Edwards KR, Malhi H, Schmidt K, Davis AR, Homad LJ, Warner NL, Chhan CB, Scharffenberger SC, Gaffney K, Hinkley T, Potchen NB, Wang JY, Price J, McElrath MJ, Olson J, King NP, Lund JM, Moodie Z, Erasmus JH, McGuire AT.
  (2024) NPJ Vaccines. 9(1):120 | doi:10.1038/s41541-024-00907-y Abstract
  Epstein-Barr virus (EBV) is associated with several malignancies, neurodegenerative disorders and is the causative agent of infectious mononucleosis. A vaccine that prevents EBV-driven morbidity and mortality remains an unmet need. EBV is orally transmitted, infecting both B cells and epithelial cells. Several virally encoded proteins are involved in entry. The gH/gL glycoprotein complex is essential for infectivity irrespective of cell type, while gp42 is essential for infection of B cells. gp350 promotes viral attachment by binding to CD21 or CD35 and is the most abundant glycoprotein on the virion. gH/gL, gp42 and gp350, are known targets of neutralizing antibodies and therefore relevant immunogens for vaccine development. Here, we developed and optimized the delivery of several alphavirus-derived replicon RNA (repRNA) vaccine candidates encoding gH/gL, gH/gL/gp42 or gp350 delivered by a cationic nanocarrier termed LION™. The lead candidate, encoding full-length gH/gL, elicited high titers of neutralizing antibodies that persisted for at least 8 months and a vaccine-specific CD8 T cell response. Transfer of vaccine-elicited IgG protected humanized mice from EBV-driven tumor formation and death following high-dose viral challenge. These data demonstrate that LION/repRNA-gH/gL is an ideal candidate vaccine for preventing EBV infection and/or related malignancies in humans.
• Growing Glycans in Rosetta: Accurate de novo glycan modeling, density fitting, and rational sequon design
  Adolf-Bryfogle J, Labonte JW, Kraft JC, Shapovalov M, Raemisch S, Lütteke T, DiMaio F, Bahl CD, Pallesen J, King NP, Gray JJ, Kulp DW, Schief WR.
  (2024) PLoS Comput Biol. 20(6):e1011895 | doi:10.1371/journal.pcbi.1011895 Abstract
  Carbohydrates and glycoproteins modulate key biological functions. However, experimental structure determination of sugar polymers is notoriously difficult. Computational approaches can aid in carbohydrate structure prediction, structure determination, and design. In this work, we developed a glycan-modeling algorithm, GlycanTreeModeler, that computationally builds glycans layer-by-layer, using adaptive kernel density estimates (KDE) of common glycan conformations derived from data in the Protein Data Bank (PDB) and from quantum mechanics (QM) calculations. GlycanTreeModeler was benchmarked on a test set of glycan structures of varying lengths, or "trees". Structures predicted by GlycanTreeModeler agreed with native structures at high accuracy for both de novo modeling and experimental density-guided building. We employed these tools to design de novo glycan trees into a protein nanoparticle vaccine to shield regions of the scaffold from antibody recognition, and experimentally verified shielding. This work will inform glycoprotein model prediction, glycan masking, and further aid computational methods in experimental structure determination and refinement.
• Computational design of non-porous pH-responsive antibody nanoparticles
  Matdes Nanoparticles Lab-led
  Yang EC, Divine R, Miranda MC, Borst AJ, Sheffler W, Zhang JZ, Decarreau J, Saragovi A, Abedi M, Goldbach N, Ahlrichs M, Dobbins C, Hand A, Cheng S, Lamb M, Levine PM, Chan S, Skotheim R, Fallas J, Ueda G, Lubner J, Somiya M, Khmelinskaia A, King NP, Baker D.
  (2024) Nat Struct Mol Biol. 31(9):1404-1412 | doi:10.1038/s41594-024-01288-5 PDF Abstract
  Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and is important for targeted delivery of biologics. Here we describe the design of octahedral non-porous nanoparticles with a targeting antibody on the two-fold symmetry axis, a designed trimer programmed to disassemble below a tunable pH transition point on the three-fold axis, and a designed tetramer on the four-fold symmetry axis. Designed non-covalent interfaces guide cooperative nanoparticle assembly from independently purified components, and a cryo-EM density map closely matches the computational design model. The designed nanoparticles can package protein and nucleic acid payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between 5.9 and 6.7. The ability to incorporate almost any antibody into a non-porous pH-dependent nanoparticle opens up new routes to antibody-directed targeted delivery.
• Rapid and automated design of two-component protein nanomaterials using ProteinMPNN
  Matdes MPNN Machine Learning Nanoparticles Lab-led
  de Haas RJ, Brunette N, Goodson A, Dauparas J, Yi SY, Yang EC, Dowling Q, Nguyen H, Kang A, Bera AK, Sankaran B, de Vries R, Baker D, King NP.
  (2024) Proc Natl Acad Sci U S A. 121(13):e2314646121 | doi:10.1073/pnas.2314646121 PDF Abstract
  The design of protein-protein interfaces using physics-based design methods such as Rosetta requires substantial computational resources and manual refinement by expert structural biologists. Deep learning methods promise to simplify protein-protein interface design and enable its application to a wide variety of problems by researchers from various scientific disciplines. Here, we test the ability of a deep learning method for protein sequence design, ProteinMPNN, to design two-component tetrahedral protein nanomaterials and benchmark its performance against Rosetta. ProteinMPNN had a similar success rate to Rosetta, yielding 13 new experimentally confirmed assemblies, but required orders of magnitude less computation and no manual refinement. The interfaces designed by ProteinMPNN were substantially more polar than those designed by Rosetta, which facilitated in vitro assembly of the designed nanomaterials from independently purified components. Crystal structures of several of the assemblies confirmed the accuracy of the design method at high resolution. Our results showcase the potential of deep learning-based methods to unlock the widespread application of designed protein-protein interfaces and self-assembling protein nanomaterials in biotechnology.
• Protective human monoclonal antibodies target conserved sites of vulnerability on the underside of influenza virus neuraminidase
  Lederhofer J, Tsybovsky Y, Nguyen L, Raab JE, Creanga A, Stephens T, Gillespie RA, Syeda HZ, Fisher BE, Skertic M, Yap C, Schaub AJ, Rawi R, Kwong PD, Graham BS, McDermott AB, Andrews SF, King NP, Kanekiyo M.
  (2024) Immunity. 57(3):574-586.e7 | doi:10.1016/j.immuni.2024.02.003 Abstract
  Continuously evolving influenza viruses cause seasonal epidemics and pose global pandemic threats. Although viral neuraminidase (NA) is an effective drug and vaccine target, our understanding of the NA antigenic landscape still remains incomplete. Here, we describe NA-specific human antibodies that target the underside of the NA globular head domain, inhibit viral propagation of a wide range of human H3N2, swine-origin variant H3N2, and H2N2 viruses, and confer both pre- and post-exposure protection against lethal H3N2 infection in mice. Cryo-EM structures of two such antibodies in complex with NA reveal non-overlapping epitopes covering the underside of the NA head. These sites are highly conserved among N2 NAs yet inaccessible unless the NA head tilts or dissociates. Our findings help guide the development of effective countermeasures against ever-changing influenza viruses by identifying hidden conserved sites of vulnerability on the NA underside.
• Macromolecular Cargo Encapsulation via In Vitro Assembly of Two-Component Protein Nanoparticles
  Hybrid materials Lab-led Nanoparticles
  Herpoldt KL, López CL, Sappington I, Pham MN, Srinivasan S, Netland J, Montgomery KS, Roy D, Prossnitz AN, Ellis D, Wargacki AJ, Pepper M, Convertine AJ, Stayton PS, King NP.
  (2024) Adv Healthc Mater. 13(11):e2303910 | doi:10.1002/adhm.202303910 Abstract
  Self-assembling protein nanoparticles are a promising class of materials for targeted drug delivery. Here, the use of a computationally designed, two-component, icosahedral protein nanoparticle is reported to encapsulate multiple macromolecular cargoes via simple and controlled self-assembly in vitro. Single-stranded RNA molecules between 200 and 2500 nucleotides in length are encapsulated and protected from enzymatic degradation for up to a month with length-dependent decay rates. Immunogenicity studies of nanoparticles packaging synthetic polymers carrying a small-molecule TLR7/8 agonist show that co-delivery of antigen and adjuvant results in a more than 20-fold increase in humoral immune responses while minimizing systemic cytokine secretion associated with free adjuvant. Coupled with the precise control over nanoparticle structure offered by computational design, robust and versatile encapsulation via in vitro assembly opens the door to a new generation of cargo-loaded protein nanoparticles that can combine the therapeutic effects of multiple drug classes.

2023

• Combinatorial immune refocusing within the influenza hemagglutinin RBD improves cross-neutralizing antibody responses
  Vaccines Nanoparticles Lab-led
  Dosey A, Ellis D, Boyoglu-Barnum S, Syeda H, Saunders M, Watson MJ, Kraft JC, Pham MN, Guttman M, Lee KK, Kanekiyo M, King NP.
  (2023) Cell Rep. 42(12):113553 | doi:10.1016/j.celrep.2023.113553 PDF Abstract
  The receptor-binding domain (RBD) of influenza virus hemagglutinin (HA) elicits potently neutralizing yet mostly strain-specific antibodies. Here, we evaluate the ability of several immunofocusing techniques to enhance the functional breadth of vaccine-elicited immune responses against the HA RBD. We present a series of "trihead" nanoparticle immunogens that display native-like closed trimeric RBDs from the HAs of several H1N1 influenza viruses. The series includes hyperglycosylated and hypervariable variants that incorporate natural and designed sequence diversity at key positions in the receptor-binding site periphery. Nanoparticle immunogens displaying triheads or hyperglycosylated triheads elicit higher hemagglutination inhibition (HAI) and neutralizing activity than the corresponding immunogens lacking either trimer-stabilizing mutations or hyperglycosylation. By contrast, mosaic nanoparticle display and antigen hypervariation do not significantly alter the magnitude or breadth of vaccine-elicited antibodies. Our results yield important insights into antibody responses against the RBD and the ability of several structure-based immunofocusing techniques to influence vaccine-elicited antibody responses.
• Antigen spacing on protein nanoparticles influences antibody responses to vaccination
  Vaccines Vaccines Nanoparticles Lab-led
  Ellis D, Dosey A, Boyoglu-Barnum S, Park YJ, Gillespie R, Syeda H, Hutchinson GB, Tsybovsky Y, Murphy M, Pettie D, Matheson N, Chan S, Ueda G, Fallas JA, Carter L, Graham BS, Veesler D, Kanekiyo M, King NP.
  (2023) Cell Rep. 42(12):113552 | doi:10.1016/j.celrep.2023.113552 PDF Abstract
  Immunogen design approaches aim to control the specificity and quality of antibody responses elicited by next-generation vaccines. Here, we use computational protein design to generate a nanoparticle vaccine platform based on the receptor-binding domain (RBD) of influenza hemagglutinin (HA) that enables precise control of antigen conformation and spacing. HA RBDs are presented as either monomers or native-like closed trimers that are connected to the underlying nanoparticle by a rigid linker that is modularly extended to precisely control antigen spacing. Nanoparticle immunogens with decreased spacing between trimeric RBDs elicit antibodies with improved hemagglutination inhibition and neutralization potency as well as binding breadth across diverse H1 HAs. Our "trihead" nanoparticle immunogen platform provides insights into anti-HA immunity, establishes antigen spacing as an important parameter in structure-based vaccine design, and embodies several design features that could be used in next-generation vaccines against influenza and other viruses.
• Broad receptor tropism and immunogenicity of a clade 3 sarbecovirus
  Lee J, Zepeda SK, Park YJ, Taylor AL, Quispe J, Stewart C, Leaf EM, Treichel C, Corti D, King NP, Starr TN, Veesler D.
  (2023) Cell Host Microbe. 31(12):1961-1973.e11 | doi:10.1016/j.chom.2023.10.018 Abstract
  Although Rhinolophus bats harbor diverse clade 3 sarbecoviruses, the structural determinants of receptor tropism along with the antigenicity of their spike (S) glycoproteins remain uncharacterized. Here, we show that the African Rhinolophus bat clade 3 sarbecovirus PRD-0038 S has a broad angiotensin-converting enzyme 2 (ACE2) usage and that receptor-binding domain (RBD) mutations further expand receptor promiscuity and enable human ACE2 utilization. We determine a cryo-EM structure of the PRD-0038 RBD bound to Rhinolophus alcyone ACE2, explaining receptor tropism and highlighting differences with SARS-CoV-1 and SARS-CoV-2. Characterization of PRD-0038 S using cryo-EM and monoclonal antibody reactivity reveals its distinct antigenicity relative to SARS-CoV-2 and identifies PRD-0038 cross-neutralizing antibodies for pandemic preparedness. PRD-0038 S vaccination elicits greater titers of antibodies cross-reacting with vaccine-mismatched clade 2 and clade 1a sarbecoviruses compared with SARS-CoV-2 S due to broader antigenic targeting, motivating the inclusion of clade 3 antigens in next-generation vaccines for enhanced resilience to viral evolution.
• In vivo selection of synthetic nucleocapsids for tissue targeting
  Hybrid materials Miniproteins Minibinders Synthetic Nucleocapsid Library Selection Lab-led
  Olshefsky A, Benasutti H, Sylvestre M, Butterfield GL, Rocklin GJ, Richardson C, Hicks DR, Lajoie MJ, Song K, Leaf E, Treichel C, Decarreau J, Ke S, Kher G, Carter L, Chamberlain JS, Baker D, King NP, Pun SH.
  (2023) Proc Natl Acad Sci U S A. 120(46):e2306129120 | doi:10.1073/pnas.2306129120 PDF Abstract
  Controlling the biodistribution of protein- and nanoparticle-based therapeutic formulations remains challenging. In vivo library selection is an effective method for identifying constructs that exhibit desired distribution behavior; library variants can be selected based on their ability to localize to the tissue or compartment of interest despite complex physiological challenges. Here, we describe further development of an in vivo library selection platform based on self-assembling protein nanoparticles encapsulating their own mRNA genomes (synthetic nucleocapsids or synNCs). We tested two distinct libraries: a low-diversity library composed of synNC surface mutations (45 variants) and a high-diversity library composed of synNCs displaying miniproteins with binder-like properties (6.2 million variants). While we did not identify any variants from the low-diversity surface library that yielded therapeutically relevant changes in biodistribution, the high-diversity miniprotein display library yielded variants that shifted accumulation toward lungs or muscles in just two rounds of in vivo selection. Our approach should contribute to achieving specific tissue homing patterns and identifying targeting ligands for diseases of interest.
• Nanoparticle display of prefusion coronavirus spike elicits S1-focused cross-reactive antibody response against diverse coronavirus subgenera
  Hutchinson GB, Abiona OM, Ziwawo CT, Werner AP, Ellis D, Tsybovsky Y, Leist SR, Palandjian C, West A, Fritch EJ, Wang N, Wrapp D, Boyoglu-Barnum S, Ueda G, Baker D, Kanekiyo M, McLellan JS, Baric RS, King NP, Graham BS, Corbett-Helaire KS.
  (2023) Nat Commun. 14(1):6195 | doi:10.1038/s41467-023-41661-4 Abstract
  Multivalent antigen display is a fast-growing area of interest toward broadly protective vaccines. Current nanoparticle-based vaccine candidates demonstrate the ability to confer antibody-mediated immunity against divergent strains of notably mutable viruses. In coronaviruses, this work is predominantly aimed at targeting conserved epitopes of the receptor binding domain. However, targeting conserved non-RBD epitopes could limit the potential for antigenic escape. To explore new potential targets, we engineered protein nanoparticles displaying coronavirus prefusion-stabilized spike (CoV_S-2P) trimers derived from MERS-CoV, SARS-CoV-1, SARS-CoV-2, hCoV-HKU1, and hCoV-OC43 and assessed their immunogenicity in female mice. Monotypic SARS-1 nanoparticles elicit cross-neutralizing antibodies against MERS-CoV and protect against MERS-CoV challenge. MERS and SARS nanoparticles elicit S1-focused antibodies, revealing a conserved site on the S N-terminal domain. Moreover, mosaic nanoparticles co-displaying distinct CoV_S-2P trimers elicit antibody responses to distant cross-group antigens and protect male and female mice against MERS-CoV challenge. Our findings will inform further efforts toward the development of pan-coronavirus vaccines.
• Multivalent antigen display on nanoparticle immunogens increases B cell clonotype diversity and neutralization breadth to pneumoviruses
  Ols S, Lenart K, Arcoverde Cerveira R, Miranda MC, Brunette N, Kochmann J, Corcoran M, Skotheim R, Philomin A, Cagigi A, Fiala B, Wrenn S, Marcandalli J, Hellgren F, Thompson EA, Lin A, Gegenfurtner F, Kumar A, Chen M, Phad GE, Graham BS, Perez L, Borst AJ, Karlsson Hedestam GB, Ruckwardt TJ, King NP, Loré K.
  (2023) Immunity. 56(10):2425-2441.e14 | doi:10.1016/j.immuni.2023.08.011 Abstract
  Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as a promising avenue for enhancing B cell responses to protein subunit vaccines. Here, we evaluated B cell responses in rhesus macaques immunized with prefusion-stabilized respiratory syncytial virus (RSV) F glycoprotein trimer compared with nanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent display skews antibody specificities and drives epitope-focusing of responding B cells. Antibody cloning and repertoire sequencing revealed that focusing was driven by the expansion of clonally distinct B cells through recruitment of diverse precursors. We identified two antibody lineages that developed either ultrapotent neutralization or pneumovirus cross-neutralization from precursor B cells with low initial affinity for the RSV-F immunogen. This suggests that increased avidity by multivalent display facilitates the activation and recruitment of these cells. Diversification of the B cell response by multivalent nanoparticle immunogens has broad implications for vaccine design.
• De novo designed ice-binding proteins from twist-constrained helices
  de Haas RJ, Tas RP, van den Broek D, Zheng C, Nguyen H, Kang A, Bera AK, King NP, Voets IK, de Vries R.
  (2023) Proc Natl Acad Sci U S A. 120(27):e2220380120 | doi:10.1073/pnas.2220380120 Abstract
  Attaining molecular-level control over solidification processes is a crucial aspect of materials science. To control ice formation, organisms have evolved bewildering arrays of ice-binding proteins (IBPs), but these have poorly understood structure-activity relationships. We propose that reverse engineering using de novo computational protein design can shed light on structure-activity relationships of IBPs. We hypothesized that the model alpha-helical winter flounder antifreeze protein uses an unusual undertwisting of its alpha-helix to align its putative ice-binding threonine residues in exactly the same direction. We test this hypothesis by designing a series of straight three-helix bundles with an ice-binding helix projecting threonines and two supporting helices constraining the twist of the ice-binding helix. Our findings show that ice-recrystallization inhibition by the designed proteins increases with the degree of designed undertwisting, thus validating our hypothesis, and opening up avenues for the computational design of IBPs.
• Broad and Durable Humoral Responses Following Single Hydrogel Immunization of SARS-CoV-2 Subunit Vaccine
  Ou BS, Saouaf OM, Yan J, Bruun TUJ, Baillet J, Zhou X, King NP, Appel EA.
  (2023) Adv Healthc Mater. 12(28):e2301495 | doi:10.1002/adhm.202301495 Abstract
  Most vaccines require several immunizations to induce robust immunity, and indeed, most SARS-CoV-2 vaccines require an initial two-shot regimen followed by several boosters to maintain efficacy. Such a complex series of immunizations unfortunately increases the cost and complexity of populations-scale vaccination and reduces overall compliance and vaccination rate. In a rapidly evolving pandemic affected by the spread of immune-escaping variants, there is an urgent need to develop vaccines capable of providing robust and durable immunity. In this work, a single immunization SARS-CoV-2 subunit vaccine is developed that can rapidly generate potent, broad, and durable humoral immunity. Injectable polymer-nanoparticle (PNP) hydrogels are leveraged as a depot technology for the sustained delivery of a nanoparticle antigen (RND-NP) displaying multiple copies of the SARS-CoV-2 receptor-binding domain (RBD) and potent adjuvants including CpG and 3M-052. Compared to a clinically relevant prime-boost regimen with soluble vaccines formulated with CpG/alum or 3M-052/alum adjuvants, PNP hydrogel vaccines more rapidly generated higher, broader, and more durable antibody responses. Additionally, these single-immunization hydrogel-based vaccines elicit potent and consistent neutralizing responses. Overall, it is shown that PNP hydrogels elicit improved anti-COVID immune responses with only a single administration, demonstrating their potential as critical technologies to enhance overall pandemic readiness.
• Fast and versatile sequence-independent protein docking for nanomaterials design using RPXDock
  Methods
  Sheffler W, Yang EC, Dowling Q, Hsia Y, Fries CN, Stanislaw J, Langowski MD, Brandys M, Li Z, Skotheim R, Borst AJ, Khmelinskaia A, King NP, Baker D.
  (2023) PLoS Comput Biol. 19(5):e1010680 | doi:10.1371/journal.pcbi.1010680 PDF Abstract
  Computationally designed multi-subunit assemblies have shown considerable promise for a variety of applications, including a new generation of potent vaccines. One of the major routes to such materials is rigid body sequence-independent docking of cyclic oligomers into architectures with point group or lattice symmetries. Current methods for docking and designing such assemblies are tailored to specific classes of symmetry and are difficult to modify for novel applications. Here we describe RPXDock, a fast, flexible, and modular software package for sequence-independent rigid-body protein docking across a wide range of symmetric architectures that is easily customizable for further development. RPXDock uses an efficient hierarchical search and a residue-pair transform (RPX) scoring method to rapidly search through multidimensional docking space. We describe the structure of the software, provide practical guidelines for its use, and describe the available functionalities including a variety of score functions and filtering tools that can be used to guide and refine docking results towards desired configurations.
• Broadly neutralizing antibodies against sarbecoviruses generated by immunization of macaques with an AS03-adjuvanted COVID-19 vaccine
  Feng Y, Yuan M, Powers JM, Hu M, Munt JE, Arunachalam PS, Leist SR, Bellusci L, Kim J, Sprouse KR, Adams LE, Sundaramurthy S, Zhu X, Shirreff LM, Mallory ML, Scobey TD, Moreno A, O'Hagan DT, Kleanthous H, Villinger FJ, Veesler D, King NP, Suthar MS, Khurana S, Baric RS, Wilson IA, Pulendran B.
  (2023) Sci Transl Med. 15(695):eadg7404 | doi:10.1126/scitranslmed.adg7404 Abstract
  The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that evade immunity elicited by vaccination has placed an imperative on the development of countermeasures that provide broad protection against SARS-CoV-2 and related sarbecoviruses. Here, we identified extremely potent monoclonal antibodies (mAbs) that neutralized multiple sarbecoviruses from macaques vaccinated with AS03-adjuvanted monovalent subunit vaccines. Longitudinal analysis revealed progressive accumulation of somatic mutation in the immunoglobulin genes of antigen-specific memory B cells (MBCs) for at least 1 year after primary vaccination. Antibodies generated from these antigen-specific MBCs at 5 to 12 months after vaccination displayed greater potency and breadth relative to those identified at 1.4 months. Fifteen of the 338 (about 4.4%) antibodies isolated at 1.4 to 6 months after the primary vaccination showed potency against SARS-CoV-2 BA.1, despite the absence of serum BA.1 neutralization. 25F9 and 20A7 neutralized authentic clade 1 sarbecoviruses (SARS-CoV, WIV-1, SHC014, SARS-CoV-2 D614G, BA.1, and Pangolin-GD) and vesicular stomatitis virus-pseudotyped clade 3 sarbecoviruses (BtKY72 and PRD-0038). 20A7 and 27A12 showed potent neutralization against all SARS-CoV-2 variants and multiple Omicron sublineages, including BA.1, BA.2, BA.3, BA.4/5, BQ.1, BQ.1.1, and XBB. Crystallography studies revealed the molecular basis of broad and potent neutralization through targeting conserved sites within the RBD. Prophylactic protection of 25F9, 20A7, and 27A12 was confirmed in mice, and administration of 25F9 particularly provided complete protection against SARS-CoV-2, BA.1, SARS-CoV, and SHC014 challenge. These data underscore the extremely potent and broad activity of these mAbs against sarbecoviruses.
• Top-down design of protein architectures with reinforcement learning
  Vaccines
  Lutz ID, Wang S, Norn C, Courbet A, Borst AJ, Zhao YT, Dosey A, Cao L, Xu J, Leaf EM, Treichel C, Litvicov P, Li Z, Goodson AD, Rivera-Sánchez P, Bratovianu AM, Baek M, King NP, Ruohola-Baker H, Baker D.
  (2023) Science. 380(6642):266-273 | doi:10.1126/science.adf6591 PDF Abstract
  As a result of evolutionary selection, the subunits of naturally occurring protein assemblies often fit together with substantial shape complementarity to generate architectures optimal for function in a manner not achievable by current design approaches. We describe a "top-down" reinforcement learning-based design approach that solves this problem using Monte Carlo tree search to sample protein conformers in the context of an overall architecture and specified functional constraints. Cryo-electron microscopy structures of the designed disk-shaped nanopores and ultracompact icosahedra are very close to the computational models. The icosohedra enable very-high-density display of immunogens and signaling molecules, which potentiates vaccine response and angiogenesis induction. Our approach enables the top-down design of complex protein nanomaterials with desired system properties and demonstrates the power of reinforcement learning in protein design.
• Progress in vaccine development for infectious diseases-a Keystone Symposia report
  Cable J, Graham BS, Koup RA, Seder RA, Karikó K, Pardi N, Barouch DH, Sharma B, Rauch S, Nachbagauer R, Forsell MNE, Schotsaert M, Ellebedy AH, Loré K, Irvine DJ, Pilkington E, Tahtinen S, Thompson EA, Feraoun Y, King NP, Saunders K, Alter G, Moin SM, Sliepen K, Karlsson Hedestam GB, Wardemann H, Pulendran B, Doria-Rose NA, He WT, Juno JA, Ataca S, Wheatley AK, McLellan JS, Walker LM, Lederhofer J, Lindesmith LC, Wille H, Hotez PJ, Bekker LG.
  (2023) Ann N Y Acad Sci. 1524(1):65-86 | doi:10.1111/nyas.14975 Abstract
  The COVID-19 pandemic has taught us many things, among the most important of which is that vaccines are one of the cornerstones of public health that help make modern longevity possible. While several different vaccines have been successful at stemming the morbidity and mortality associated with various infectious diseases, many pathogens/diseases remain recalcitrant to the development of effective vaccination. Recent advances in vaccine technology, immunology, structural biology, and other fields may yet yield insight that will address these diseases; they may also help improve societies' preparedness for future pandemics. On June 1-4, 2022, experts in vaccinology from academia, industry, and government convened for the Keystone symposium "Progress in Vaccine Development for Infectious Diseases" to discuss state-of-the-art technologies, recent advancements in understanding vaccine-mediated immunity, and new aspects of antigen design to aid vaccine effectiveness.
• Process development of a SARS-CoV-2 nanoparticle vaccine
  Martinez-Cano D, Ravichandran R, Le H, Wong HE, Jagannathan B, Liu EJ, Bailey W, Yang J, Matthies K, Barkhordarian H, Shah B, Srinivasan N, Zhang J, Hsu A, Wypych J, Stevens J, Piedmonte DM, Miranda LP, Carter L, Murphy M, King NP, Soice N. | doi:10.1016/j.procbio.2023.03.014 Abstract
  One of the outcomes from the global COVID-19 pandemic caused by SARS-CoV-2 has been an acceleration of development timelines to provide treatments in a timely manner. For example, it has recently been demonstrated that the development of monoclonal antibody therapeutics from vector construction to IND submission can be achieved in five to six months rather than the traditional ten-to-twelve-month timeline using CHO cells [1], [2]. This timeline is predicated on leveraging existing, robust platforms for upstream and downstream processes, analytical methods, and formulation. These platforms also reduce; the requirement for ancillary studies such as cell line stability, or long-term product stability studies. Timeline duration was further reduced by employing a transient cell line for early material supply and using a stable cell pool to manufacture toxicology study materials. The development of non-antibody biologics utilizing traditional biomanufacturing processes in CHO cells within a similar timeline presents additional challenges, such as the lack of platform processes and additional analytical assay development. In this manuscript, we describe the rapid development of a robust and reproducible process for a two-component self-assembling protein nanoparticle vaccine for SARS-CoV-2. Our work has demonstrated a successful academia-industry partnership model that responded to the COVID-19 global pandemic quickly and efficiently and could improve our preparedness for future pandemic threats.
• Improving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains
  Matdes Nanoparticles Lab-led
  Wang JYJ, Khmelinskaia A, Sheffler W, Miranda MC, Antanasijevic A, Borst AJ, Torres SV, Shu C, Hsia Y, Nattermann U, Ellis D, Walkey C, Ahlrichs M, Chan S, Kang A, Nguyen H, Sydeman C, Sankaran B, Wu M, Bera AK, Carter L, Fiala B, Murphy M, Baker D, Ward AB, King NP.
  (2023) Proc Natl Acad Sci U S A. 120(11):e2214556120 | doi:10.1073/pnas.2214556120 PDF Abstract
  Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice, they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. The retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the nanoparticles we describe may be broadly useful in biotechnological applications.
• Design and immunological evaluation of two-component protein nanoparticle vaccines for East Coast fever
  Vaccines Nanoparticles Lab-led
  Lacasta A, Kim HC, Kepl E, Gachogo R, Chege N, Ojuok R, Muriuki C, Mwalimu S, Touboul G, Stiber A, Poole EJ, Ndiwa N, Fiala B, King NP, Nene V. | doi:10.3389/fimmu.2022.1015840 Abstract
  Nanoparticle vaccines usually prime stronger immune responses than soluble antigens. Within this class of subunit vaccines, the recent development of computationally designed self-assembling two-component protein nanoparticle scaffolds provides a powerful and versatile platform for displaying multiple copies of one or more antigens. Here we report the generation of three different nanoparticle immunogens displaying 60 copies of p67C, an 80 amino acid polypeptide from a candidate vaccine antigen of , and their immunogenicity in cattle. p67C is a truncation of p67, the major surface protein of the sporozoite stage of , an apicomplexan parasite that causes an often-fatal bovine disease called East Coast fever (ECF) in sub-Saharan Africa. Compared to I32-19 and I32-28, we found that I53-50 nanoparticle scaffolds displaying p67C had the best biophysical characteristics. p67C-I53-50 also outperformed the other two nanoparticles in stimulating p67C-specific IgG1 and IgG2 antibodies and CD4 T-cell responses, as well as sporozoite neutralizing capacity. In experimental cattle vaccine trials, p67C-I53-50 induced significant immunity to ECF, suggesting that the I53-50 scaffold is a promising candidate for developing novel nanoparticle vaccines. To our knowledge this is the first application of computationally designed nanoparticles to the development of livestock vaccines.
• Computational design of constitutively active cGAS
  Enzymes Lab-led
  Dowling QM, Volkman HE, Gray EE, Ovchinnikov S, Cambier S, Bera AK, Sankaran B, Johnson MR, Bick MJ, Kang A, Stetson DB, King NP.
  (2023) Nat Struct Mol Biol. 30(1):72-80 | doi:10.1038/s41594-022-00862-z Abstract
  Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor critical for the innate immune response to intracellular pathogens, DNA damage, tumorigenesis and senescence. Binding to double-stranded DNA (dsDNA) induces conformational changes in cGAS that activate the enzyme to produce 2'-3' cyclic GMP-AMP (cGAMP), a second messenger that initiates a potent interferon (IFN) response through its receptor, STING. Here, we combined two-state computational design with informatics-guided design to create constitutively active, dsDNA ligand-independent cGAS (CA-cGAS). We identified CA-cGAS mutants with IFN-stimulating activity approaching that of dsDNA-stimulated wild-type cGAS. DNA-independent adoption of the active conformation was directly confirmed by X-ray crystallography. In vivo expression of CA-cGAS in tumor cells resulted in STING-dependent tumor regression, demonstrating that the designed proteins have therapeutically relevant biological activity. Our work provides a general framework for stabilizing active conformations of enzymes and provides CA-cGAS variants that could be useful as genetically encoded adjuvants and tools for understanding inflammatory diseases.

2022

• Induction of cross-neutralizing antibodies by a permuted hepatitis C virus glycoprotein nanoparticle vaccine candidate
  Sliepen K, Radić L, Capella-Pujol J, Watanabe Y, Zon I, Chumbe A, Lee WH, de Gast M, Koopsen J, Koekkoek S, Del Moral-Sánchez I, Brouwer PJM, Ravichandran R, Ozorowski G, King NP, Ward AB, van Gils MJ, Crispin M, Schinkel J, Sanders RW.
  (2022) Nat Commun. 13(1):7271 | doi:10.1038/s41467-022-34961-8 Abstract
  Hepatitis C virus (HCV) infection affects approximately 58 million people and causes ~300,000 deaths yearly. The only target for HCV neutralizing antibodies is the highly sequence diverse E1E2 glycoprotein. Eliciting broadly neutralizing antibodies that recognize conserved cross-neutralizing epitopes is important for an effective HCV vaccine. However, most recombinant HCV glycoprotein vaccines, which usually include only E2, induce only weak neutralizing antibody responses. Here, we describe recombinant soluble E1E2 immunogens that were generated by permutation of the E1 and E2 subunits. We displayed the E2E1 immunogens on two-component nanoparticles and these nanoparticles induce significantly more potent neutralizing antibody responses than E2. Next, we generated mosaic nanoparticles co-displaying six different E2E1 immunogens. These mosaic E2E1 nanoparticles elicit significantly improved neutralization compared to monovalent E2E1 nanoparticles. These results provide a roadmap for the generation of an HCV vaccine that induces potent and broad neutralization.
• Co-display of diverse spike proteins on nanoparticles broadens sarbecovirus neutralizing antibody responses
  Brinkkemper M, Veth TS, Brouwer PJM, Turner H, Poniman M, Burger JA, Bouhuijs JH, Olijhoek W, Bontjer I, Snitselaar JL, Caniels TG, van der Linden CA, Ravichandran R, Villaudy J, van der Velden YU, Sliepen K, van Gils MJ, Ward AB, King NP, Heck AJR, Sanders RW.
  (2022) iScience. 25(12):105649 | doi:10.1016/j.isci.2022.105649 Abstract
  The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses continuous challenges in combating the virus. Here, we describe vaccination strategies to broaden SARS-CoV-2 and sarbecovirus immunity by combining spike proteins based on different viruses or viral strains displayed on two-component protein nanoparticles. First, we combined spike proteins based on ancestral and Beta SARS-CoV-2 strains to broaden SARS-CoV-2 immune responses. Inclusion of Beta spike improved neutralizing antibody responses against SARS-CoV-2 Beta, Gamma, and Omicron BA.1 and BA.4/5. A third vaccination with ancestral SARS-CoV-2 spike also improved cross-neutralizing antibody responses against SARS-CoV-2 variants, in particular against the Omicron sublineages. Second, we combined SARS-CoV and SARS-CoV-2 spike proteins to broaden sarbecovirus immune responses. Adding SARS-CoV spike to a SARS-CoV-2 spike vaccine improved neutralizing responses against SARS-CoV and SARS-like bat sarbecoviruses SHC014 and WIV1. These results should inform the development of broadly active SARS-CoV-2 and pan-sarbecovirus vaccines and highlight the versatility of two-component nanoparticles for displaying diverse antigens.
• Antigen- and scaffold-specific antibody responses to protein nanoparticle immunogens
  Vaccines Nanoparticles Lab-led
  Kraft JC, Pham MN, Shehata L, Brinkkemper M, Boyoglu-Barnum S, Sprouse KR, Walls AC, Cheng S, Murphy M, Pettie D, Ahlrichs M, Sydeman C, Johnson M, Blackstone A, Ellis D, Ravichandran R, Fiala B, Wrenn S, Miranda M, Sliepen K, Brouwer PJM, Antanasijevic A, Veesler D, Ward AB, Kanekiyo M, Pepper M, Sanders RW, King NP.
  (2022) Cell Rep Med. 3(10):100780 | doi:10.1016/j.xcrm.2022.100780 Abstract
  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.
• Correction: IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS-CoV-2
  Hale M, Netland J, Chen Y, Thouvenel CD, Smith KN, Rich LM, Vanderwall ER, Miranda MC, Eggenberger J, Hao L, Watson MJ, Mundorff CC, Rodda LB, King NP, Guttman M, Gale M, Abraham J, Debley JS, Pepper M, Rawlings DJ. | doi:10.1084/jem.2022084908172022c
• Durable protection against the SARS-CoV-2 Omicron variant is induced by an adjuvanted subunit vaccine
  Arunachalam PS, Feng Y, Ashraf U, Hu M, Walls AC, Edara VV, Zarnitsyna VI, Aye PP, Golden N, Miranda MC, Green KWM, Threeton BM, Maness NJ, Beddingfield BJ, Bohm RP, Scheuermann SE, Goff K, Dufour J, Russell-Lodrigue K, Kepl E, Fiala B, Wrenn S, Ravichandran R, Ellis D, Carter L, Rogers K, Shirreff LM, Ferrell DE, Deb Adhikary NR, Fontenot J, Hammond HL, Frieman M, Grifoni A, Sette A, O'Hagan DT, Van Der Most R, Rappuoli R, Villinger F, Kleanthous H, Rappaport J, Suthar MS, Veesler D, Wang TT, King NP, Pulendran B.
  (2022) Sci Transl Med. 14(658):eabq4130 | doi:10.1126/scitranslmed.abq4130 Abstract
  Despite the remarkable efficacy of COVID-19 vaccines, waning immunity and the emergence of SARS-CoV-2 variants such as Omicron represents a global health challenge. Here, we present data from a study in nonhuman primates demonstrating durable protection against the Omicron BA.1 variant induced by a subunit SARS-CoV-2 vaccine comprising the receptor binding domain of the ancestral strain (RBD-Wu) on the I53-50 nanoparticle adjuvanted with AS03, which was recently authorized for use in individuals 18 years or older. Vaccination induced neutralizing antibody (nAb) titers that were maintained at high concentrations for at least 1 year after two doses, with a pseudovirus nAb geometric mean titer (GMT) of 1978 and a live virus nAb GMT of 1331 against the ancestral strain but not against the Omicron BA.1 variant. However, a booster dose at 6 to 12 months with RBD-Wu or RBD-β (RBD from the Beta variant) displayed on I53-50 elicited high neutralizing titers against the ancestral and Omicron variants. In addition, we observed persistent neutralization titers against a panel of sarbecoviruses, including SARS-CoV. Furthermore, there were substantial and persistent memory T and B cell responses reactive to Beta and Omicron variants. Vaccination resulted in protection against Omicron infection in the lung and suppression of viral burden in the nares at 6 weeks after the final booster immunization. Even at 6 months after vaccination, we observed protection in the lung and rapid control of virus in the nares. These results highlight the durable and cross-protective immunity elicited by the AS03-adjuvanted RBD-I53-50 nanoparticle vaccine.
• Vaccination with a structure-based stabilized version of malarial antigen Pfs48/45 elicits ultra-potent transmission-blocking antibody responses
  McLeod B, Mabrouk MT, Miura K, Ravichandran R, Kephart S, Hailemariam S, Pham TP, Semesi A, Kucharska I, Kundu P, Huang WC, Johnson M, Blackstone A, Pettie D, Murphy M, Kraft JC, Leaf EM, Jiao Y, van de Vegte-Bolmer M, van Gemert GJ, Ramjith J, King CR, MacGill RS, Wu Y, Lee KK, Jore MM, King NP, Lovell JF, Julien JP.
  (2022) Immunity. 55(9):1680-1692.e8 | doi:10.1016/j.immuni.2022.07.015 Abstract
  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.
• Distinct sensitivities to SARS-CoV-2 variants in vaccinated humans and mice
  Walls AC, VanBlargan LA, Wu K, Choi A, Navarro MJ, Lee D, Avena L, Berrueta DM, Pham MN, Elbashir S, Kraft JC, Miranda MC, Kepl E, Johnson M, Blackstone A, Sprouse K, Fiala B, O'Connor MA, Brunette N, Arunachalam PS, Shirreff L, Rogers K, Carter L, Fuller DH, Villinger F, Pulendran B, Diamond MS, Edwards DK, King NP, Veesler D.
  (2022) Cell Rep. 40(9):111299 | doi:10.1016/j.celrep.2022.111299 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 (VOCs) 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 VOCs. We report that vaccinated BALB/c mice do not recapitulate faithfully the breadth and potency of neutralizing antibody responses elicited by various vaccine platforms against VOCs, compared with non-human primates or humans, suggesting caution should be exercised when interpreting data obtained with this animal model.
• IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS-CoV-2
  Hale M, Netland J, Chen Y, Thouvenel CD, Smith KN, Rich LM, Vanderwall ER, Miranda MC, Eggenberger J, Hao L, Watson MJ, Mundorff CC, Rodda LB, King NP, Guttman M, Gale M, Abraham J, Debley JS, Pepper M, Rawlings DJ. | doi:10.1084/jem.20220849 Abstract
  Humoral immunity to SARS-CoV-2 can be supplemented with polyclonal sera from convalescent donors or an engineered monoclonal antibody (mAb) product. While pentameric IgM antibodies are responsible for much of convalescent sera's neutralizing capacity, all available mAbs are based on the monomeric IgG antibody subtype. We now show that IgM mAbs derived from immune memory B cell receptors are potent neutralizers of SARS-CoV-2. IgM mAbs outperformed clonally identical IgG antibodies across a range of affinities and SARS-CoV-2 receptor-binding domain epitopes. Strikingly, efficacy against SARS-CoV-2 viral variants was retained for IgM but not for clonally identical IgG. To investigate the biological role for IgM memory in SARS-CoV-2, we also generated IgM mAbs from antigen-experienced IgM+ memory B cells in convalescent donors, identifying a potent neutralizing antibody. Our results highlight the therapeutic potential of IgM mAbs and inform our understanding of the role for IgM memory against a rapidly mutating pathogen.
• Increasing computational protein design literacy through cohort-based learning for undergraduate students
  doi:https://pubs.acs.org/doi/10.1021/acs.jchemed.2c00500 Abstract
  Rigorous undergraduate research experiences are essential for improving student success in graduate education and STEM careers. During the COVID-19 pandemic, undergraduate researchers in our institution lost their work-study positions due to a pause of in-person research activities. Losing their work-study positions posed a great financial burden on the students, and eliminated their opportunity to experience undergraduate research. To address the need for new research opportunities, we created a paid, fully-remote, and cohort-based computational research curriculum. This research curriculum used previously-developed protein design methods as a platform to educate and train undergraduate student researchers. In this program, students learned computational design methods to modulate the stability of previously-characterized, designed protein assemblies. Their results uncovered gaps in the accuracy of current nanomaterial assembly design algorithms. Our program provides a model for other educational organizations with access to basic computing infrastructure to offer structured research training opportunities to a larger number of undergraduate researchers than is typically possible in a wet lab environment.
• Safety and immunogenicity of a SARS-CoV-2 recombinant protein nanoparticle vaccine (GBP510) adjuvanted with AS03: A randomised, placebo-controlled, observer-blinded phase 1/2 trial
  Song JY, Choi WS, Heo JY, Lee JS, Jung DS, Kim SW, Park KH, Eom JS, Jeong SJ, Lee J, Kwon KT, Choi HJ, Sohn JW, Kim YK, Noh JY, Kim WJ, Roman F, Ceregido MA, Solmi F, Philippot A, Walls AC, Carter L, Veesler D, King NP, Kim H, Ryu JH, Lee SJ, Park YW, Park HK, Cheong HJ. | doi:10.1016/j.eclinm.2022.101569 Abstract
  Vaccination has helped to mitigate the COVID-19 pandemic. Ten traditional and novel vaccines have been listed by the World Health Organization for emergency use. Additional alternative approaches may better address ongoing vaccination globally, where there remains an inequity in vaccine distribution. GBP510 is a recombinant protein vaccine, which consists of self-assembling, two-component nanoparticles, displaying the receptor-binding domain (RBD) in a highly immunogenic array.
• Immunization with a self-assembling nanoparticle vaccine displaying EBV gH/gL protects humanized mice against lethal viral challenge
  Malhi H, Homad LJ, Wan YH, Poudel B, Fiala B, Borst AJ, Wang JY, Walkey C, Price J, Wall A, Singh S, Moodie Z, Carter L, Handa S, Correnti CE, Stoddard BL, Veesler D, Pancera M, Olson J, King NP, McGuire AT.
  (2022) Cell Rep Med. 3(6):100658 | doi:10.1016/j.xcrm.2022.100658 Abstract
  Epstein-Barr virus (EBV) is a cancer-associated pathogen responsible for 165,000 deaths annually. EBV is also the etiological agent of infectious mononucleosis and is linked to multiple sclerosis and rheumatoid arthritis. Thus, an EBV vaccine would have a significant global health impact. EBV is orally transmitted and has tropism for epithelial and B cells. Therefore, a vaccine would need to prevent infection of both in the oral cavity. Passive transfer of monoclonal antibodies against the gH/gL glycoprotein complex prevent experimental EBV infection in humanized mice and rhesus macaques, suggesting that gH/gL is an attractive vaccine candidate. Here, we evaluate the immunogenicity of several gH/gL nanoparticle vaccines. All display superior immunogenicity relative to monomeric gH/gL. A nanoparticle displaying 60 copies of gH/gL elicits antibodies that protect against lethal EBV challenge in humanized mice, whereas antibodies elicited by monomeric gH/gL do not. These data motivate further development of gH/gL nanoparticle vaccines for EBV.
• Structure, receptor recognition, and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein
  Vaccines
  Tortorici MA, Walls AC, Joshi A, Park YJ, Eguia RT, Miranda MC, Kepl E, Dosey A, Stevens-Ayers T, Boeckh MJ, Telenti A, Lanzavecchia A, King NP, Corti D, Bloom JD, Veesler D.
  (2022) Cell. 185(13):2279-2291.e17 | doi:10.1016/j.cell.2022.05.019 Abstract
  The isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. We determined the structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and showed that its domain 0 recognizes sialosides. We identified that the CCoV-HuPn-2018 spike binds canine, feline, and porcine aminopeptidase N (APN) orthologs, which serve as entry receptors, and determined the structure of the receptor-binding B domain in complex with canine APN. The introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single-nucleotide polymorphisms might account for viral detection in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 spike-mediated entry, underscoring the cross-neutralizing activity among ɑ-coronaviruses. These data pave the way for vaccine and therapeutic development targeting this zoonotic pathogen representing the eighth human-infecting coronavirus.
• Adjuvanting a subunit SARS-CoV-2 vaccine with clinically relevant adjuvants induces durable protection in mice
  Grigoryan L, Lee A, Walls AC, Lai L, Franco B, Arunachalam PS, Feng Y, Luo W, Vanderheiden A, Floyd K, Wrenn S, Pettie D, Miranda MC, Kepl E, Ravichandran R, Sydeman C, Brunette N, Murphy M, Fiala B, Carter L, Coffman RL, Novack D, Kleanthous H, O'Hagan DT, van der Most R, McLellan JS, Suthar M, Veesler D, King NP, Pulendran B.
  (2022) NPJ Vaccines. 7(1):55 | doi:10.1038/s41541-022-00472-2 Abstract
  Adjuvants enhance the magnitude and the durability of the immune response to vaccines. However, there is a paucity of comparative studies on the nature of the immune responses stimulated by leading adjuvant candidates. In this study, we compared five clinically relevant adjuvants in mice-alum, AS03 (a squalene-based adjuvant supplemented with α-tocopherol), AS37 (a TLR7 ligand emulsified in alum), CpG1018 (a TLR9 ligand emulsified in alum), O/W 1849101 (a squalene-based adjuvant)-for their capacity to stimulate immune responses when combined with a subunit vaccine under clinical development. We found that all four of the adjuvant candidates surpassed alum with respect to their capacity to induce enhanced and durable antigen-specific antibody responses. The TLR-agonist-based adjuvants CpG1018 (TLR9) and AS37 (TLR7) induced Th1-skewed CD4+ T cell responses, while alum, O/W, and AS03 induced a balanced Th1/Th2 response. Consistent with this, adjuvants induced distinct patterns of early innate responses. Finally, vaccines adjuvanted with AS03, AS37, and CpG1018/alum-induced durable neutralizing-antibody responses and significant protection against the B.1.351 variant 7 months following immunization. These results, together with our recent results from an identical study in non-human primates (NHPs), provide a comparative benchmarking of five clinically relevant vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV-2 subunit vaccines to provide durable protection against the B.1.351 variant. Furthermore, these results reveal differences between the widely-used C57BL/6 mouse strain and NHP animal models, highlighting the importance of species selection for future vaccine and adjuvant studies.
• Epitope-focused immunogen design based on the ebolavirus glycoprotein HR2-MPER region
  Schoeder CT, Gilchuk P, Sangha AK, Ledwitch KV, Malherbe DC, Zhang X, Binshtein E, Williamson LE, Martina CE, Dong J, Armstrong E, Sutton R, Nargi R, Rodriguez J, Kuzmina N, Fiala B, King NP, Bukreyev A, Crowe JE, Meiler J.
  (2022) PLoS Pathog. 18(5):e1010518 | doi:10.1371/journal.ppat.1010518 Abstract
  The three human pathogenic ebolaviruses: Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) virus, cause severe disease with high fatality rates. Epitopes of ebolavirus glycoprotein (GP) recognized by antibodies with binding breadth for all three ebolaviruses are of major interest for rational vaccine design. In particular, the heptad repeat 2 -membrane-proximal external region (HR2-MPER) epitope is relatively conserved between EBOV, BDBV, and SUDV GP and targeted by human broadly-neutralizing antibodies. To study whether this epitope can serve as an immunogen for the elicitation of broadly-reactive antibody responses, protein design in Rosetta was employed to transplant the HR2-MPER epitope identified from a co-crystal structure with the known broadly-reactive monoclonal antibody (mAb) BDBV223 onto smaller scaffold proteins. From computational analysis, selected immunogen designs were produced as recombinant proteins and functionally validated, leading to the identification of a sterile alpha motif (SAM) domain displaying the BDBV-HR2-MPER epitope near its C terminus as a promising candidate. The immunogen was fused to one component of a self-assembling, two-component nanoparticle and tested for immunogenicity in rabbits. Robust titers of cross-reactive serum antibodies to BDBV and EBOV GPs and moderate titers to SUDV GP were induced following immunization. To confirm the structural composition of the immunogens, solution NMR studies were conducted and revealed structural flexibility in the C-terminal residues of the epitope. Overall, our study represents the first report on an epitope-focused immunogen design based on the structurally challenging BDBV-HR2-MPER epitope.
• Engineering Self-Assembling Protein Nanoparticles for Therapeutic Delivery
  Olshefsky A, Richardson C, Pun SH, King NP.
  (2022) Bioconjug Chem. 33(11):2018-2034 | doi:10.1021/acs.bioconjchem.2c00030 Abstract
  Despite remarkable advances over the past several decades, many therapeutic nanomaterials fail to overcome major in vivo delivery barriers. Controlling immunogenicity, optimizing biodistribution, and engineering environmental responsiveness are key outstanding delivery problems for most nanotherapeutics. However, notable exceptions exist including some lipid and polymeric nanoparticles, some virus-based nanoparticles, and nanoparticle vaccines where immunogenicity is desired. Self-assembling protein nanoparticles offer a powerful blend of modularity and precise designability to the field, and have the potential to solve many of the major barriers to delivery. In this review, we provide a brief overview of key designable features of protein nanoparticles and their implications for therapeutic delivery applications. We anticipate that protein nanoparticles will rapidly grow in their prevalence and impact as clinically relevant delivery platforms.
• Computational design of mechanically coupled axle-rotor protein assemblies
  Courbet A, Hansen J, Hsia Y, Bethel N, Park YJ, Xu C, Moyer A, Boyken SE, Ueda G, Nattermann U, Nagarajan D, Silva DA, Sheffler W, Quispe J, Nord A, King N, Bradley P, Veesler D, Kollman J, Baker D.
  (2022) Science. 376(6591):383-390 | doi:10.1126/science.abm1183 PDF Abstract
  Natural molecular machines contain protein components that undergo motion relative to each other. Designing such mechanically constrained nanoscale protein architectures with internal degrees of freedom is an outstanding challenge for computational protein design. Here we explore the de novo construction of protein machinery from designed axle and rotor components with internal cyclic or dihedral symmetry. We find that the axle-rotor systems assemble in vitro and in vivo as designed. Using cryo-electron microscopy, we find that these systems populate conformationally variable relative orientations reflecting the symmetry of the coupled components and the computationally designed interface energy landscape. These mechanical systems with internal degrees of freedom are a step toward the design of genetically encodable nanomachines.
• Structure-based design of stabilized recombinant influenza neuraminidase tetramers
  Vaccines Antigen Design Lab-led
  Ellis D, Lederhofer J, Acton OJ, Tsybovsky Y, Kephart S, Yap C, Gillespie RA, Creanga A, Olshefsky A, Stephens T, Pettie D, Murphy M, Sydeman C, Ahlrichs M, Chan S, Borst AJ, Park YJ, Lee KK, Graham BS, Veesler D, King NP, Kanekiyo M.
  (2022) Nat Commun. 13(1):1825 | doi:10.1038/s41467-022-29416-z Abstract
  Influenza virus neuraminidase (NA) is a major antiviral drug target and has recently reemerged as a key target of antibody-mediated protective immunity. Here we show that recombinant NAs across non-bat subtypes adopt various tetrameric conformations, including an "open" state that may help explain poorly understood variations in NA stability across viral strains and subtypes. We use homology-directed protein design to uncover the structural principles underlying these distinct tetrameric conformations and stabilize multiple recombinant NAs in the "closed" state, yielding two near-atomic resolution structures of NA by cryo-EM. In addition to enhancing thermal stability, conformational stabilization improves affinity to protective antibodies elicited by viral infection, including antibodies targeting a quaternary epitope and the broadly conserved catalytic site. Stabilized NAs can also be integrated into viruses without affecting fitness. Our findings provide a deeper understanding of NA structure, stability, and antigenicity, and establish design strategies for reinforcing the conformational integrity of recombinant NA proteins.
• Mannose-binding lectin and complement mediate follicular localization and enhanced immunogenicity of diverse protein nanoparticle immunogens
  Read BJ, Won L, Kraft JC, Sappington I, Aung A, Wu S, Bals J, Chen C, Lee KK, Lingwood D, King NP, Irvine DJ.
  (2022) Cell Rep. 38(2):110217 | doi:10.1016/j.celrep.2021.110217 Abstract
  Nanoparticle (NP) vaccine formulations promote immune responses through multiple mechanisms. We recently reported that mannose-binding lectin (MBL) triggers trafficking of glycosylated HIV Env-immunogen NPs to lymph node follicles. Here, we investigate effects of MBL and complement on NP forms of HIV and other viral antigens. MBL recognition of oligomannose on gp120 nanoparticles significantly increases antigen accumulation in lymph nodes and antigen-specific germinal center (GC) responses. MBL and complement also mediate follicular trafficking and enhance GC responses to influenza, HBV, and HPV particulate antigens. Using model protein nanoparticles bearing titrated levels of glycosylation, we determine that mannose patches at a minimal density of 2.1 × 10 mannose patches/nm are required to trigger follicular targeting, which increases with increasing glycan density up to at least ∼8.2 × 10 patches/nm. Thus, innate immune recognition of glycans has a significant impact on humoral immunity, and these findings provide a framework for engineering glycan recognition to optimize vaccine efficacy.

2021

• Airway antibodies emerge according to COVID-19 severity and wane rapidly but reappear after SARS-CoV-2 vaccination
  Cagigi A, Yu M, Österberg B, Svensson J, Falck-Jones S, Vangeti S, Åhlberg E, Azizmohammadi L, Warnqvist A, Falck-Jones R, Gubisch PC, Ödemis M, Ghafoor F, Eisele M, Lenart K, Bell M, Johansson N, Albert J, Sälde J, Pettie DD, Murphy MP, Carter L, King NP, Ols S, Normark J, Ahlm C, Forsell MN, Färnert A, Loré K, Smed-Sörensen A. | doi:10.1172/jci.insight.151463 Abstract
  Understanding the presence and durability of antibodies against SARS-CoV-2 in the airways is required to provide insights into the ability of individuals to neutralize the virus locally and prevent viral spread. Here, we longitudinally assessed both systemic and airway immune responses upon SARS-CoV-2 infection in a clinically well-characterized cohort of 147 infected individuals representing the full spectrum of COVID-19 severity, from asymptomatic infection to fatal disease. In addition, we evaluated how SARS-CoV-2 vaccination influenced the antibody responses in a subset of these individuals during convalescence as compared with naive individuals. Not only systemic but also airway antibody responses correlated with the degree of COVID-19 disease severity. However, although systemic IgG levels were durable for up to 8 months, airway IgG and IgA declined significantly within 3 months. After vaccination, there was an increase in both systemic and airway antibodies, in particular IgG, often exceeding the levels found during acute disease. In contrast, naive individuals showed low airway antibodies after vaccination. In the former COVID-19 patients, airway antibody levels were significantly elevated after the boost vaccination, highlighting the importance of prime and boost vaccinations for previously infected individuals to obtain optimal mucosal protection.
• Limited access to antigen drives generation of early B cell memory while restraining the plasmablast response
  Glaros V, Rauschmeier R, Artemov AV, Reinhardt A, Ols S, Emmanouilidi A, Gustafsson C, You Y, Mirabello C, Björklund ÅK, Perez L, King NP, Månsson R, Angeletti D, Loré K, Adameyko I, Busslinger M, Kreslavsky T.
  (2021) Immunity. 54(9):2005-2023.e10 | doi:10.1016/j.immuni.2021.08.017 Abstract
  Cell fate decisions during early B cell activation determine the outcome of responses to pathogens and vaccines. We examined the early B cell response to T-dependent antigen in mice by single-cell RNA sequencing. Early after immunization, a homogeneous population of activated precursors (APs) gave rise to a transient wave of plasmablasts (PBs), followed a day later by the emergence of germinal center B cells (GCBCs). Most APs rapidly exited the cell cycle, giving rise to non-GC-derived early memory B cells (eMBCs) that retained an AP-like transcriptional profile. Rapid decline of antigen availability controlled these events; provision of excess antigen precluded cell cycle exit and induced a new wave of PBs. Fate mapping revealed a prominent contribution of eMBCs to the MBC pool. Quiescent cells with an MBC phenotype dominated the early response to immunization in primates. A reservoir of APs/eMBCs may enable rapid readjustment of the immune response when failure to contain a threat is manifested by increased antigen availability.
• Multimeric antibodies from antigen-specific human IgM+ memory B cells restrict Plasmodium parasites
  Thouvenel CD, Fontana MF, Netland J, Krishnamurty AT, Takehara KK, Chen Y, Singh S, Miura K, Keitany GJ, Lynch EM, Portugal S, Miranda MC, King NP, Kollman JM, Crompton PD, Long CA, Pancera M, Rawlings DJ, Pepper M. | doi:10.1084/jem.20200942 Abstract
  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.
• Qualification of ELISA and neutralization methodologies to measure SARS-CoV-2 humoral immunity using human clinical samples
  Larsen SE, Berube BJ, Pecor T, Cross E, Brown BP, Williams BD, Johnson E, Qu P, Carter L, Wrenn S, Kepl E, Sydeman C, King NP, Baldwin SL, Coler RN. | doi:10.1016/j.jim.2021.113160 Abstract
  In response to the SARS-CoV-2 pandemic many vaccines have been developed and evaluated in human clinical trials. The humoral immune response magnitude, composition and efficacy of neutralizing SARS-CoV-2 are essential endpoints for these trials. Robust assays that are reproducibly precise, linear, and specific for SARS-CoV-2 antigens would be beneficial for the vaccine pipeline. In this work we describe the methodologies and clinical qualification of three SARS-CoV-2 endpoint assays. We developed and qualified Endpoint titer ELISAs for total IgG, IgG1, IgG3, IgG4, IgM and IgA to evaluate the magnitude of specific responses to the trimeric spike (S) antigen and total IgG specific to the spike receptor binding domain (RBD) of SARS-CoV-2. We also qualified a pseudovirus neutralization assay which evaluates functional antibody titers capable of inhibiting the entry and replication of a lentivirus containing the Spike antigen of SARS-CoV-2. To complete the suite of assays we qualified a plaque reduction neutralization test (PRNT) methodology using the 2019-nCoV/USA-WA1/2020 isolate of SARS-CoV-2 to assess neutralizing titers of antibodies in plasma from normal healthy donors and convalescent COVID-19 individuals.
• Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice
  Dalvie NC, Rodriguez-Aponte SA, Hartwell BL, Tostanoski LH, Biedermann AM, Crowell LE, Kaur K, Kumru OS, Carter L, Yu J, Chang A, McMahan K, Courant T, Lebas C, Lemnios AA, Rodrigues KA, Silva M, Johnston RS, Naranjo CA, Tracey MK, Brady JR, Whittaker CA, Yun D, Brunette N, Wang JY, Walkey C, Fiala B, Kar S, Porto M, Lok M, Andersen H, Lewis MG, Love KR, Camp DL, Silverman JM, Kleanthous H, Joshi SB, Volkin DB, Dubois PM, Collin N, King NP, Barouch DH, Irvine DJ, Love JC. | doi:10.1073/pnas.2106845118 Abstract
  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 cost. 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.
• Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines
  Vaccines Nanoparticles Lab-led
  Walls AC, Miranda MC, Schäfer A, Pham MN, Greaney A, Arunachalam PS, Navarro MJ, Tortorici MA, Rogers K, O'Connor MA, Shirreff L, Ferrell DE, Bowen J, Brunette N, Kepl E, Zepeda SK, Starr T, Hsieh CL, Fiala B, Wrenn S, Pettie D, Sydeman C, Sprouse KR, Johnson M, Blackstone A, Ravichandran R, Ogohara C, Carter L, Tilles SW, Rappuoli R, Leist SR, Martinez DR, Clark M, Tisch R, O'Hagan DT, Van Der Most R, Van Voorhis WC, Corti D, McLellan JS, Kleanthous H, Sheahan TP, Smith KD, Fuller DH, Villinger F, Bloom J, Pulendran B, Baric RS, King NP, Veesler D.
  (2021) Cell. 184(21):5432-5447.e16 | doi:10.1016/j.cell.2021.09.015 Abstract
  Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.
• Hallmarks of icosahedral virus capsids emerged during laboratory evolution of a bacterial enzyme
  Olshefsky A, King NP.
  (2021) Trends Biochem Sci. 46(11):863-865 | doi:10.1016/j.tibs.2021.08.001 Abstract
  Viruses are fascinating molecular machines that inspire many therapeutic design efforts. Tetter, Terasaka, Steinauer et al. recently reported the laboratory evolution of a synthetic nucleocapsid from a bacterial enzyme. Their work sheds light on the potential origin of viruses and points the way to improved nanotechnology platforms.
• Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM
  Antanasijevic A, Sewall LM, Cottrell CA, Carnathan DG, Jimenez LE, Ngo JT, Silverman JB, Groschel B, Georgeson E, Bhiman J, Bastidas R, LaBranche C, Allen JD, Copps J, Perrett HR, Rantalainen K, Cannac F, Yang YR, de la Peña AT, Rocha RF, Berndsen ZT, Baker D, King NP, Sanders RW, Moore JP, Crotty S, Crispin M, Montefiori DC, Burton DR, Schief WR, Silvestri G, Ward AB.
  (2021) Nat Commun. 12(1):4817 | doi:10.1038/s41467-021-25087-4 Abstract
  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.
• Stabilization of the SARS-CoV-2 Spike Receptor-Binding Domain Using Deep Mutational Scanning and Structure-Based Design
  Vaccines Antigen Design Nanoparticles Lab-led
  Ellis D, Brunette N, Crawford KHD, Walls AC, Pham MN, Chen C, Herpoldt KL, Fiala B, Murphy M, Pettie D, Kraft JC, Malone KD, Navarro MJ, Ogohara C, Kepl E, Ravichandran R, Sydeman C, Ahlrichs M, Johnson M, Blackstone A, Carter L, Starr TN, Greaney AJ, Lee KK, Veesler D, Bloom JD, King NP. | doi:10.3389/fimmu.2021.710263 Abstract
  The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.
• Adjuvanting a subunit COVID-19 vaccine to induce protective immunity
  Vaccines
  Arunachalam PS, Walls AC, Golden N, Atyeo C, Fischinger S, Li C, Aye P, Navarro MJ, Lai L, Edara VV, Röltgen K, Rogers K, Shirreff L, Ferrell DE, Wrenn S, Pettie D, Kraft JC, Miranda MC, Kepl E, Sydeman C, Brunette N, Murphy M, Fiala B, Carter L, White AG, Trisal M, Hsieh CL, Russell-Lodrigue K, Monjure C, Dufour J, Spencer S, Doyle-Meyers L, Bohm RP, Maness NJ, Roy C, Plante JA, Plante KS, Zhu A, Gorman MJ, Shin S, Shen X, Fontenot J, Gupta S, O'Hagan DT, Van Der Most R, Rappuoli R, Coffman RL, Novack D, McLellan JS, Subramaniam S, Montefiori D, Boyd SD, Flynn JL, Alter G, Villinger F, Kleanthous H, Rappaport J, Suthar MS, King NP, Veesler D, Pulendran B.
  (2021) Nature. 594(7862):253-258 | doi:10.1038/s41586-021-03530-2 Abstract
  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 protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).
• Designed proteins assemble antibodies into modular nanocages
  Divine R, Dang HV, Ueda G, Fallas JA, Vulovic I, Sheffler W, Saini S, Zhao YT, Raj IX, Morawski PA, Jennewein MF, Homad LJ, Wan YH, Tooley MR, Seeger F, Etemadi A, Fahning ML, Lazarovits J, Roederer A, Walls AC, Stewart L, Mazloomi M, King NP, Campbell DJ, McGuire AT, Stamatatos L, Ruohola-Baker H, Mathieu J, Veesler D, Baker D. | doi:10.1126/science.abd9994 Abstract
  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.
• Structure-based design of novel polyhedral protein nanomaterials
  Matdes Nanoparticles Lab-led
  Khmelinskaia A, Wargacki A, King NP. | doi:10.1016/j.mib.2021.03.003 Abstract
  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.
• Quadrivalent influenza nanoparticle vaccines induce broad protection
  Vaccines Nanoparticles Lab-led
  Boyoglu-Barnum S, Ellis D, Gillespie RA, Hutchinson GB, Park YJ, Moin SM, Acton OJ, Ravichandran R, Murphy M, Pettie D, Matheson N, Carter L, Creanga A, Watson MJ, Kephart S, Ataca S, Vaile JR, Ueda G, Crank MC, Stewart L, Lee KK, Guttman M, Baker D, Mascola JR, Veesler D, Graham BS, King NP, Kanekiyo M.
  (2021) Nature. 592(7855):623-628 | doi:10.1038/s41586-021-03365-x Abstract
  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.
• Dynamics of Neutralizing Antibody Titers in the Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection
  Crawford KHD, Dingens AS, Eguia R, Wolf CR, Wilcox N, Logue JK, Shuey K, Casto AM, Fiala B, Wrenn S, Pettie D, King NP, Greninger AL, Chu HY, Bloom JD.
  (2021) J Infect Dis. 223(2):197-205 | doi:10.1093/infdis/jiaa618 Abstract
  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.
• Complete and cooperative in vitro assembly of computationally designed self-assembling protein nanomaterials
  Matdes Nanoparticles Lab-led
  Wargacki AJ, Wörner TP, van de Waterbeemd M, Ellis D, Heck AJR, King NP.
  (2021) Nat Commun. 12(1):883 | doi:10.1038/s41467-021-21251-y Abstract
  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.
• Immunofocusing and enhancing autologous Tier-2 HIV-1 neutralization by displaying Env trimers on two-component protein nanoparticles
  Vaccines
  Brouwer PJM, Antanasijevic A, de Gast M, Allen JD, Bijl TPL, Yasmeen A, Ravichandran R, Burger JA, Ozorowski G, Torres JL, LaBranche C, Montefiori DC, Ringe RP, van Gils MJ, Moore JP, Klasse PJ, Crispin M, King NP, Ward AB, Sanders RW.
  (2021) NPJ Vaccines. 6(1):24 | doi:10.1038/s41541-021-00285-9 Abstract
  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.
• Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection
  Vaccines
  Brouwer PJM, Brinkkemper M, Maisonnasse P, Dereuddre-Bosquet N, Grobben M, Claireaux M, de Gast M, Marlin R, Chesnais V, Diry S, Allen JD, Watanabe Y, Giezen JM, Kerster G, Turner HL, van der Straten K, van der Linden CA, Aldon Y, Naninck T, Bontjer I, Burger JA, Poniman M, Mykytyn AZ, Okba NMA, Schermer EE, van Breemen MJ, Ravichandran R, Caniels TG, van Schooten J, Kahlaoui N, Contreras V, Lemaître J, Chapon C, Fang RHT, Villaudy J, Sliepen K, van der Velden YU, Haagmans BL, de Bree GJ, Ginoux E, Ward AB, Crispin M, King NP, van der Werf S, van Gils MJ, Le Grand R, Sanders RW.
  (2021) Cell. 184(5):1188-1200.e19 | doi:10.1016/j.cell.2021.01.035 Abstract
  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.

2020

• Aldehyde Oxidase Contributes to All--Retinoic Acid Biosynthesis in Human Liver
  Zhong G, Seaman CJ, Paragas EM, Xi H, Herpoldt KL, King NP, Jones JP, Isoherranen N.
  (2021) Drug Metab Dispos. 49(3):202-211 | doi:10.1124/dmd.120.000296 Abstract
  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.
• Designed proteins assemble antibodies into modular nanocages
  Divine R, Dang HV, Ueda G, Fallas JA, Vulovic I, Sheffler W, Saini S, Zhao YT, Raj IX, Morawski PA, Jennewein MF, Homad LJ, Wan YH, Tooley MR, Seeger F, Etemadi A, Fahning ML, Lazarovits J, Roederer A, Walls AC, Stewart L, Mazloomi M, King NP, Campbell DJ, McGuire AT, Stamatatos L, Ruohola-Baker H, Mathieu J, Veesler D, Baker D. | doi:10.1101/2020.12.01.406611 Abstract
  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.
• Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19
  Rodda LB, Netland J, Shehata L, Pruner KB, Morawski PA, Thouvenel CD, Takehara KK, Eggenberger J, Hemann EA, Waterman HR, Fahning ML, Chen Y, Hale M, Rathe J, Stokes C, Wrenn S, Fiala B, Carter L, Hamerman JA, King NP, Gale M, Campbell DJ, Rawlings DJ, Pepper M.
  (2021) Cell. 184(1):169-183.e17 | doi:10.1016/j.cell.2020.11.029 Abstract
  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.
• Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2
  Vaccines Lab-led Nanoparticles
  Walls AC, Fiala B, Schäfer A, Wrenn S, Pham MN, Murphy M, Tse LV, Shehata L, O'Connor MA, Chen C, Navarro MJ, Miranda MC, Pettie D, Ravichandran R, Kraft JC, Ogohara C, Palser A, Chalk S, Lee EC, Guerriero K, Kepl E, Chow CM, Sydeman C, Hodge EA, Brown B, Fuller JT, Dinnon KH, Gralinski LE, Leist SR, Gully KL, Lewis TB, Guttman M, Chu HY, Lee KK, Fuller DH, Baric RS, Kellam P, Carter L, Pepper M, Sheahan TP, Veesler D, King NP.
  (2020) Cell. 183(5):1367-1382.e17 | doi:10.1016/j.cell.2020.10.043 Abstract
  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.
• Dynamics of neutralizing antibody titers in the months after SARS-CoV-2 infection
  doi:10.1093/infdis/jiaa618 Abstract
  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.
• A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver
  Vaccines
  Wang LT, Pereira LS, Flores-Garcia Y, O'Connor J, Flynn BJ, Schön A, Hurlburt NK, Dillon M, Yang ASP, Fabra-García A, Idris AH, Mayer BT, Gerber MW, Gottardo R, Mason RD, Cavett N, Ballard RB, Kisalu NK, Molina-Cruz A, Nelson J, Vistein R, Barillas-Mury C, Amino R, Baker D, King NP, Sauerwein RW, Pancera M, Cockburn IA, Zavala F, Francica JR, Seder RA.
  (2020) Immunity. 53(4):733-744.e8 | doi:10.1016/j.immuni.2020.08.014 Abstract
  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.
• Serological identification of SARS-CoV-2 infections among children visiting a hospital during the initial Seattle outbreak
  Dingens AS, Crawford KHD, Adler A, Steele SL, Lacombe K, Eguia R, Amanat F, Walls AC, Wolf CR, Murphy M, Pettie D, Carter L, Qin X, King NP, Veesler D, Krammer F, Dickerson JA, Chu HY, Englund JA, Bloom JD.
  (2020) Nat Commun. 11(1):4378 | doi:10.1038/s41467-020-18178-1 Abstract
  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.
• Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens
  Antanasijevic A, Ueda G, Brouwer PJM, Copps J, Huang D, Allen JD, Cottrell CA, Yasmeen A, Sewall LM, Bontjer I, Ketas TJ, Turner HL, Berndsen ZT, Montefiori DC, Klasse PJ, Crispin M, Nemazee D, Moore JP, Sanders RW, King NP, Baker D, Ward AB.
  (2020) PLoS Pathog. 16(8):e1008665 | doi:10.1371/journal.ppat.1008665 Abstract
  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.
• Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding
  Vaccines
  Starr TN, Greaney AJ, Hilton SK, Ellis D, Crawford KHD, Dingens AS, Navarro MJ, Bowen JE, Tortorici MA, Walls AC, King NP, Veesler D, Bloom JD.
  (2020) Cell. 182(5):1295-1310.e20 | doi:10.1016/j.cell.2020.08.012 Abstract
  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.
• Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations
  Martin JT, Cottrell CA, Antanasijevic A, Carnathan DG, Cossette BJ, Enemuo CA, Gebru EH, Choe Y, Viviano F, Fischinger S, Tokatlian T, Cirelli KM, Ueda G, Copps J, Schiffner T, Menis S, Alter G, Schief WR, Crotty S, King NP, Baker D, Silvestri G, Ward AB, Irvine DJ.
  (2020) NPJ Vaccines. 5(1):72 | doi:10.1038/s41541-020-00223-1 Abstract
  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.
• Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens
  Vaccines Nanoparticles
  Ueda G, Antanasijevic A, Fallas JA, Sheffler W, Copps J, Ellis D, Hutchinson GB, Moyer A, Yasmeen A, Tsybovsky Y, Park YJ, Bick MJ, Sankaran B, Gillespie RA, Brouwer PJ, Zwart PH, Veesler D, Kanekiyo M, Graham BS, Sanders RW, Moore JP, Klasse PJ, Ward AB, King NP, Baker D. | doi:10.7554/eLife.57659 Abstract
  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.
• Serological identification of SARS-CoV-2 infections among children visiting a hospital during the initial Seattle outbreak
  Dingens AS, Crawford KHD, Adler A, Steele SL, Lacombe K, Eguia R, Amanat F, Walls AC, Wolf CR, Murphy M, Pettie D, Carter L, Qin X, King NP, Veesler D, Krammer F, Dickerson JA, Chu HY, Englund JA, Bloom JD. | doi:10.1101/2020.05.26.20114124 Abstract
  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 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 had 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.
• Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays
  Crawford KHD, Eguia R, Dingens AS, Loes AN, Malone KD, Wolf CR, Chu HY, Tortorici MA, Veesler D, Murphy M, Pettie D, King NP, Balazs AB, Bloom JD. | doi:10.3390/v12050513 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 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.
• New Vaccine Design and Delivery Technologies
  Vaccines
  Kanekiyo M, Ellis D, King NP.
  (2019) J Infect Dis. 219(Suppl_1):S88-S96 | doi:10.1093/infdis/jiy745 Abstract
  Technological advances in immunology, protein design, and genetic delivery have unlocked new possibilities for vaccine concepts and delivery technologies that were previously inaccessible. These next-generation vaccine design efforts are particularly promising in their potential to provide solutions to challenging targets for which conventional approaches have proven ineffective-for example, a universal influenza vaccine. In this perspective, we discuss emerging approaches to vaccine design and engineering based on recent insights into immunology, structural biology, computational biology, and immunoengineering. We anticipate that these cutting-edge, interdisciplinary approaches will lead to breakthrough vaccine concepts for ever-evolving and (re)emerging influenza viruses, with important ramifications for global public health.

2019

• Design and structure of two new protein cages illustrate successes and ongoing challenges in protein engineering
  Cannon KA, Park RU, Boyken SE, Nattermann U, Yi S, Baker D, King NP, Yeates TO.
  (2020) Protein Sci. 29(4):919-929 | doi:10.1002/pro.3802 Abstract
  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.
• De novo design of tunable, pH-driven conformational changes
  Boyken SE, Benhaim MA, Busch F, Jia M, Bick MJ, Choi H, Klima JC, Chen Z, Walkey C, Mileant A, Sahasrabuddhe A, Wei KY, Hodge EA, Byron S, Quijano-Rubio A, Sankaran B, King NP, Lippincott-Schwartz J, Wysocki VH, Lee KK, Baker D.
  (2019) Science. 364(6441):658-664 | doi:10.1126/science.aav7897 Abstract
  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.
• Enhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle
  Brouwer PJM, Antanasijevic A, Berndsen Z, Yasmeen A, Fiala B, Bijl TPL, Bontjer I, Bale JB, Sheffler W, Allen JD, Schorcht A, Burger JA, Camacho M, Ellis D, Cottrell CA, Behrens AJ, Catalano M, Del Moral-Sánchez I, Ketas TJ, LaBranche C, van Gils MJ, Sliepen K, Stewart LJ, Crispin M, Montefiori DC, Baker D, Moore JP, Klasse PJ, Ward AB, King NP, Sanders RW.
  (2019) Nat Commun. 10(1):4272 | doi:10.1038/s41467-019-12080-1 Abstract
  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.
• Multimerization of an Alcohol Dehydrogenase by Fusion to a Designed Self-Assembling Protein Results in Enhanced Bioelectrocatalytic Operational Stability
  Bulutoglu B, Macazo FC, Bale J, King N, Baker D, Minteer SD, Banta S.
  (2019) ACS Appl Mater Interfaces. 11(22):20022-20028 | doi:10.1021/acsami.9b04256 Abstract
  Proteins designed for supramolecular assembly provide a simple means to immobilize and organize enzymes for biotechnology applications. We have genetically fused the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus to a computationally designed cage-forming protein (O3-33). The trimeric form of the O3-33-AdhD fusion protein was most active in solution. The immobilization of the fusion protein on bioelectrodes leads to a doubling of the electrochemical operational stability as compared to the unfused control proteins. Thus, the fusion of enzymes to the designed self-assembling domains offers a simple strategy to increase the stability in biocatalytic systems.
• Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus
  Vaccines Nanoparticles Lab-led
  Marcandalli J, Fiala B, Ols S, Perotti M, de van der Schueren W, Snijder J, Hodge E, Benhaim M, Ravichandran R, Carter L, Sheffler W, Brunner L, Lawrenz M, Dubois P, Lanzavecchia A, Sallusto F, Lee KK, Veesler D, Correnti CE, Stewart LJ, Baker D, Loré K, Perez L, King NP.
  (2019) Cell. 176(6):1420-1431.e17 | doi:10.1016/j.cell.2019.01.046 Abstract
  Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses ∼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.

2018

• Confirmation of intersubunit connectivity and topology of designed protein complexes by native MS
  Sahasrabuddhe A, Hsia Y, Busch F, Sheffler W, King NP, Baker D, Wysocki VH.
  (2018) Proc Natl Acad Sci U S A. 115(6):1268-1273 | doi:10.1073/pnas.1713646115 Abstract
  Computational protein design provides the tools to expand the diversity of protein complexes beyond those found in nature. Understanding the rules that drive proteins to interact with each other enables the design of protein-protein interactions to generate specific protein assemblies. In this work, we designed protein-protein interfaces between dimers and trimers to generate dodecameric protein assemblies with dihedral point group symmetry. We subsequently analyzed the designed protein complexes by native MS. We show that the use of ion mobility MS in combination with surface-induced dissociation (SID) allows for the rapid determination of the stoichiometry and topology of designed complexes. The information collected along with the speed of data acquisition and processing make SID ion mobility MS well-suited to determine key structural features of designed protein complexes, thereby circumventing the requirement for more time- and sample-consuming structural biology approaches.

2017

• Evolution of a designed protein assembly encapsulating its own RNA genome
  Hybrid materials Nanoparticles Synthetic Nucleocapsid Lab-led
  Butterfield GL, Lajoie MJ, Gustafson HH, Sellers DL, Nattermann U, Ellis D, Bale JB, Ke S, Lenz GH, Yehdego A, Ravichandran R, Pun SH, King NP, Baker D.
  (2017) Nature. 552(7685):415-420 | doi:10.1038/nature25157 Abstract
  The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a 'blank slate' to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to approximately 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at 'top-down' modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary 'bottom-up' approach with considerable advantages in programmability and control.

2016

• Designed proteins induce the formation of nanocage-containing extracellular vesicles
  Hybrid materials Lab-led Nanoparticles
  Votteler J, Ogohara C, Yi S, Hsia Y, Nattermann U, Belnap DM, King NP, Sundquist WI.
  (2016) Nature. 540(7632):292-295 | doi:10.1038/nature20607 Abstract
  Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids or proteins, but strategies for engineering hybrid biological materials are only beginning to emerge. Here we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as 'enveloped protein nanocages' (EPNs). Robust EPN biogenesis requires protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the endosomal sorting complexes required for transport (ESCRT) machinery. A variety of synthetic proteins with these functional elements induce EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochemical analyses and cryo-electron microscopy reveal that one design, EPN-01, comprises small (~100 nm) vesicles containing multiple protein nanocages that closely match the structure of the designed 60-subunit self-assembling scaffold. EPNs that incorporate the vesicular stomatitis viral glycoprotein can fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These results show how proteins can be programmed to direct the formation of hybrid biological materials that perform complex tasks, and establish EPNs as a class of designed, modular, genetically-encoded nanomaterials that can transfer molecules between cells.
• Multivalent Display of Antifreeze Proteins by Fusion to Self-Assembling Protein Cages Enhances Ice-Binding Activities
  Phippen SW, Stevens CA, Vance TD, King NP, Baker D, Davies PL.
  (2016) Biochemistry. 55(49):6811-6820 | doi:10.1021/acs.biochem.6b00864 Abstract
  Antifreeze proteins (AFPs) are small monomeric proteins that adsorb to the surface of ice to inhibit ice crystal growth and impart freeze resistance to the organisms producing them. Previously, monomeric AFPs have been conjugated to the termini of branched polymers to increase their activity through the simultaneous binding of more than one AFP to ice. Here, we describe a superior approach to increasing AFP activity through oligomerization that eliminates the need for conjugation reactions with varying levels of efficiency. A moderately active AFP from a fish and a hyperactive AFP from an Antarctic bacterium were genetically fused to the C-termini of one component of the 24-subunit protein cage T33-21, resulting in protein nanoparticles that multivalently display exactly 12 AFPs. The resulting nanoparticles exhibited freezing point depression >50-fold greater than that seen with the same concentration of monomeric AFP and a similar increase in the level of ice-recrystallization inhibition. These results support the anchored clathrate mechanism of binding of AFP to ice. The enhanced freezing point depression could be due to the difficulty of overgrowing a larger AFP on the ice surface and the improved ice-recrystallization inhibition to the ability of the nanoparticle to simultaneously bind multiple ice grains. Oligomerization of these proteins using self-assembling protein cages will be useful in a variety of biotechnology and cryobiology applications.
• Accurate design of megadalton-scale two-component icosahedral protein complexes
  Matdes Nanoparticles
  Bale JB, Gonen S, Liu Y, Sheffler W, Ellis D, Thomas C, Cascio D, Yeates TO, Gonen T, King NP, Baker D.
  (2016) Science. 353(6297):389-94 | doi:10.1126/science.aaf8818 Abstract
  Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.
• Design of a hyperstable 60-subunit protein dodecahedron. [corrected]
  Matdes Nanoparticles
  Hsia Y, Bale JB, Gonen S, Shi D, Sheffler W, Fong KK, Nattermann U, Xu C, Huang PS, Ravichandran R, Yi S, Davis TN, Gonen T, King NP, Baker D.
  (2016) Nature. 535(7610):136-9 | doi:10.1038/nature18010 Abstract
  The dodecahedron [corrected] is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The dodecahedron [corrected] is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

2007-2015

• Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression
  Bale JB, Park RU, Liu Y, Gonen S, Gonen T, Cascio D, King NP, Yeates TO, Baker D.
  (2015) Protein Sci. 24(10):1695-701 | doi:10.1002/pro.2748 Abstract
  We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials.
• Accurate design of co-assembling multi-component protein nanomaterials
  Matdes Nanoparticles Lab-led
  King NP, Bale JB, Sheffler W, McNamara DE, Gonen S, Gonen T, Yeates TO, Baker D.
  (2014) Nature. 510(7503):103-8 | doi:10.1038/nature13404 Abstract
  The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self-assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications.
• Practical approaches to designing novel protein assemblies
  King NP, Lai YT.
  (2013) Curr Opin Struct Biol. 23(4):632-8 | doi:10.1016/j.sbi.2013.06.002 Abstract
  Molecular self-assembly offers a means by which sophisticated materials can be constructed with unparalleled precision. Designing self-assembling protein structures is of particular interest as a result of the unique functional capabilities of proteins. Custom-designed protein materials could lead to new possibilities in therapeutics, bioenergy, and materials science. Although the field was long hampered by the challenges involved in designing such complex molecules, novel approaches and computational tools have recently led to remarkable progress. Here we review recent design studies in the context of three fundamental aspects of self-assembling materials: subunit organization, subunit interactions, and regulation of assembly.
• Computational design of self-assembling protein nanomaterials with atomic level accuracy
  Matdes Nanoparticles Lab-led
  King NP, Sheffler W, Sawaya MR, Vollmar BS, Sumida JP, André I, Gonen T, Yeates TO, Baker D.
  (2012) Science. 336(6085):1171-4 | doi:10.1126/science.1219364 Abstract
  We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.
• Principles for designing ordered protein assemblies
  Lai YT, King NP, Yeates TO.
  (2012) Trends Cell Biol. 22(12):653-61 | doi:10.1016/j.tcb.2012.08.004 Abstract
  In nature, many proteins have evolved to have self-complementary shapes. This drives them to assemble into supramolecular structures, sometimes of great complexity, and often carrying out sophisticated cellular functions. Designing novel proteins that can self-assemble into similarly complex structures is a longstanding goal in bioengineering. New ideas, combined with continually improving computer algorithms, are making it possible to advance on that goal, bringing wide-ranging applications in synthetic biology within reach. Prospective applications range from vaccine design to molecular delivery to bioactive materials. Recent strategies and examples of successfully designed protein cages, layers, and crystals are reviewed.
• Protein stabilization in a highly knotted protein polymer
  Sayre TC, Lee TM, King NP, Yeates TO.
  (2011) Protein Eng Des Sel. 24(8):627-30 | doi:10.1093/protein/gzr024 Abstract
  The polypeptide backbones of a few proteins are tied in a knot. The biophysical effects and potential biological roles of knots are not well understood. Here, we test the consequences of protein knotting by taking a monomeric protein, carbonic anhydrase II, whose native structure contains a shallow knot, and polymerizing it end-to-end to form a deeply and multiply knotted polymeric filament. Thermal stability experiments show that the polymer is stabilized against loss of structure and aggregation by the presence of deep knots.
• Structure and folding of a designed knotted protein
  King NP, Jacobitz AW, Sawaya MR, Goldschmidt L, Yeates TO.
  (2010) Proc Natl Acad Sci U S A. 107(48):20732-7 | doi:10.1073/pnas.1007602107 Abstract
  A very small number of natural proteins have folded configurations in which the polypeptide backbone is knotted. Relatively little is known about the folding energy landscapes of such proteins, or how they have evolved. We explore those questions here by designing a unique knotted protein structure. Biophysical characterization and X-ray crystal structure determination show that the designed protein folds to the intended configuration, tying itself in a knot in the process, and that it folds reversibly. The protein folds to its native, knotted configuration approximately 20 times more slowly than a control protein, which was designed to have a similar tertiary structure but to be unknotted. Preliminary kinetic experiments suggest a complicated folding mechanism, providing opportunities for further characterization. The findings illustrate a situation where a protein is able to successfully traverse a complex folding energy landscape, though the amino acid sequence of the protein has not been subjected to evolutionary pressure for that ability. The success of the design strategy--connecting two monomers of an intertwined homodimer into a single protein chain--supports a model for evolution of knotted structures via gene duplication.
• Structures and functional implications of an AMP-binding cystathionine beta-synthase domain protein from a hyperthermophilic archaeon
  King NP, Lee TM, Sawaya MR, Cascio D, Yeates TO.
  (2008) J Mol Biol. 380(1):181-92 | doi:10.1016/j.jmb.2008.04.073 Abstract
  Cystathionine beta-synthase domains are found in a myriad of proteins from organisms across the tree of life and have been hypothesized to function as regulatory modules that sense the energy charge of cells. Here we characterize the structure and stability of PAE2072, a dimeric tandem cystathionine beta-synthase domain protein from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. Crystal structures of the protein in unliganded and AMP-bound forms, determined at resolutions of 2.10 and 2.35 A, respectively, reveal remarkable conservation of key functional features seen in the gamma subunit of the eukaryotic AMP-activated protein kinase. The structures also confirm the presence of a suspected intermolecular disulfide bond between the two subunits that is shown to stabilize the protein. Our AMP-bound structure represents a first step in investigating the function of a large class of uncharacterized prokaryotic proteins. In addition, this work extends previous studies that have suggested that, in certain thermophilic microbes, disulfide bonds play a key role in stabilizing intracellular proteins and protein-protein complexes.
• Knotted and topologically complex proteins as models for studying folding and stability
  Yeates TO, Norcross TS, King NP.
  (2007) Curr Opin Chem Biol. 11(6):595-603 | doi:10.1016/j.cbpa.2007.10.002 Abstract
  Among proteins of known three-dimensional structure, only a few possess complex topological features such as knotted or interlinked (catenated) protein backbones. Such unusual proteins offer potentially unique insights into folding pathways and stabilization mechanisms. They also present special challenges for both theorists and computational scientists interested in understanding and predicting protein-folding behavior. Here, we review complex topological features in proteins with a focus on recent progress on the identification and characterization of knotted and interlinked protein systems. Also, an approach is described for designing an expanded set of knotted proteins.
• Identification of rare slipknots in proteins and their implications for stability and folding
  King NP, Yeates EO, Yeates TO.
  (2007) J Mol Biol. 373(1):153-66 | doi:10.1016/j.jmb.2007.07.042 Abstract
  Among the thousands of known three-dimensional protein folds, only a few have been found whose backbones are in knotted configurations. The rarity of knotted proteins has important implications for how natural proteins reach their natively folded states. Proteins with such unusual features offer unique opportunities for studying the relationships between structure, folding, and stability. Here we report the identification of a unique slipknot feature in the fold of a well-known thermostable protein, alkaline phosphatase. A slipknot is created when a knot is formed by part of a protein chain, after which the backbone doubles back so that the entire structure becomes unknotted in a mathematical sense. Slipknots are therefore not detected by computational tests that look for knots in complete protein structures. A computational survey looking specifically for slipknots in the Protein Data Bank reveals a few other instances in addition to alkaline phosphatase. Unexpected similarities are noted among some of the proteins identified. In addition, two transmembrane proteins are found to contain slipknots. Finally, mutagenesis experiments on alkaline phosphatase are used to probe the contribution the slipknot feature makes to thermal stability. The trends and conserved features observed in these proteins provide new insights into mechanisms of protein folding and stability.