- Wargacki AJ, Wörner TP, van de Waterbeemd M, Ellis D, Heck AJR, King NP. (2021).
Complete and cooperative in vitro assembly of computationally designed self-assembling protein nanomaterials.
Nat Commun. 02 2021. 12(1):883.
(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.
- 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).
Dynamics of Neutralizing Antibody Titers in the Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.
J Infect Dis. 02 2021. 223(2):197-205.
(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.
- 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).
Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19.
Cell. 01 2021. 184(1):169-183.e17.
(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.
- S Arunachalam P, Walls AC, Golden N, Atyeo C, Fischinger S, Li C, Aye P, Navarro MJ, Lai L, Edara VV, Roltgen 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, Doyle-Meyer L, Bohm RB, Maness NJ, Roy C, Plante JA, Plante KS, Zhu A, Gorman MJ, Shin S, Shen X, Fontenot J, Gupta S, O Hagan DT, Most RV, 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 M, King NP, Veesler D, Pulendran B. (2021).
Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates.
bioRxiv. Feb 2021. :.
(Abstract)
The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor-binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.
- 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).
Immunofocusing and enhancing autologous Tier-2 HIV-1 neutralization by displaying Env trimers on two-component protein nanoparticles.
NPJ Vaccines. Feb 2021. 6(1):24.
(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.
- 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).
Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection.
Cell. Mar 2021. 184(5):1188-1200.e19.
(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.
- Zhong G, Seaman CJ, Paragas EM, Xi H, Herpoldt KL, King NP, Jones JP, Isoherranen N. (2021).
Aldehyde Oxidase Contributes to All--Retinoic Acid Biosynthesis in Human Liver.
Drug Metab Dispos. Mar 2021. 49(3):202-211.
(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.
- 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. (2021).
Multimeric antibodies from antigen-specific human IgM+ memory B cells restrict Plasmodium parasites.
J Exp Med. Apr 2021. 218(4):.
(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.
- 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).
Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2.
Cell. 11 2020. 183(5):1367-1382.e17.
(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.
- 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).
Serological identification of SARS-CoV-2 infections among children visiting a hospital during the initial Seattle outbreak.
Nat Commun. 09 2020. 11(1):4378.
(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.
- 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).
Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding.
Cell. 09 2020. 182(5):1295-1310.e20.
(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.
- 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).
Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations.
NPJ Vaccines. 2020. 5:72.
(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.
- 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).
Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens.
PLoS Pathog. 08 2020. 16(8):e1008665.
(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.
- 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. (2020).
Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens.
Elife. 08 2020. 9:.
(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.
- 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. (2020).
Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays.
Viruses. 05 2020. 12(5):.
(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.
- Cannon KA, Park RU, Boyken SE, Nattermann U, Yi S, Baker D, King NP, Yeates TO. (2020).
Design and structure of two new protein cages illustrate successes and ongoing challenges in protein engineering.
Protein Sci. 04 2020. 29(4):919-929.
(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.
- 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).
Seroprevalence of SARS-CoV-2 among children visiting a hospital during the initial Seattle outbreak.
medRxiv. May 2020. :.
(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 nine were seropositive in April for a period seroprevalence of >1%. Most seropositive children (8/10) were not suspected of having had COVID-19. The sera of most seropositive children had neutralizing activity, including one that neutralized at a dilution >1:18,000. Therefore, among children seeking medical care, the frequency of SARS-CoV-2 infection increased markedly during the early Seattle outbreak despite few positive viral tests.
- 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).
Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations.
NPJ Vaccines. Aug 2020. 5(1):72.
(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.
- Rodda LB, Netland J, Shehata L, Pruner KB, Morawski PA, Thouvenel C, Takehara KK, Eggenberger J, Hemann E, Waterman HR, Fahning ML, Chen Y, Rathe J, Stokes C, Wrenn S, Fiala B, Carter L, Hamerman JA, King NP, Gale M, Campbell DJ, Rawlings D, Pepper M. (2020).
Functional SARS-CoV-2-specific immune memory persists after mild COVID-19.
Res Sq. Aug 2020. :.
(Abstract)
The recently emerged SARS-CoV-2 virus is currently causing a global pandemic and cases continue to rise. The majority of infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that might contribute to herd immunity. Thus, we performed a longitudinal assessment of individuals recovered from mildly symptomatic COVID-19 to determine if they develop and sustain immunological memory against the virus. We found that recovered individuals developed SARS-CoV-2-specific IgG antibody and neutralizing plasma, as well as virus-specific memory B and T cells that not only persisted, but in some cases increased numerically over three months following symptom onset. Furthermore, the SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral immunity: memory T cells secreted IFN-γ and expanded upon antigen re-encounter, while memory B cells expressed receptors capable of neutralizing virus when expressed as antibodies. These findings demonstrate that mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks associated with antiviral protective immunity.
- Rodda LB, Netland J, Shehata L, Pruner KB, Morawski PM, Thouvenel C, Takehara KK, Eggenberger J, Hemann EA, Waterman HR, Fahning ML, Chen Y, Rathe J, Stokes C, Wrenn S, Fiala B, Carter LP, Hamerman JA, King NP, Gale M, Campbell DJ, Rawlings D, Pepper M. (2020).
Functional SARS-CoV-2-specific immune memory persists after mild COVID-19.
medRxiv. Aug 2020. :.
(Abstract)
The recently emerged SARS-CoV-2 virus is currently causing a global pandemic and cases continue to rise. The majority of infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that might contribute to herd immunity. Thus, we performed a longitudinal assessment of individuals recovered from mildly symptomatic COVID-19 to determine if they develop and sustain immunological memory against the virus. We found that recovered individuals developed SARS-CoV-2-specific IgG antibody and neutralizing plasma, as well as virus-specific memory B and T cells that not only persisted, but in some cases increased numerically over three months following symptom onset. Furthermore, the SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral immunity: memory T cells secreted IFN-γ and expanded upon antigen re-encounter, while memory B cells expressed receptors capable of neutralizing virus when expressed as antibodies. These findings demonstrate that mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks associated with antiviral protective immunity.
- 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, 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).
Elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for SARS-CoV-2.
bioRxiv. Aug 2020. :.
(Abstract)
A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, we describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms for inducing potent neutralizing antibody responses and have launched cGMP manufacturing efforts to advance the lead RBD nanoparticle vaccine into the clinic.
- 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. (2020).
Dynamics of neutralizing antibody titers in the months after SARS-CoV-2 infection.
J Infect Dis. Sep 2020. :.
(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.
- 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).
A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver.
Immunity. Oct 2020. 53(4):733-744.e8.
(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.
- 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. (2020).
Designed proteins assemble antibodies into modular nanocages.
bioRxiv. Dec 2020. :.
(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.
- Votteler J, Ogohara C, Yi S, Hsia Y, Nattermann U, Belnap DM, King NP, Sundquist WI. (2016).
Designed proteins induce the formation of nanocage-containing extracellular vesicles.
Nature. 12 2016. 540(7632):292-295.
(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.
- 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).
Design of a hyperstable 60-subunit protein dodecahedron. [corrected].
Nature. 07 2016. 535(7610):136-9.
(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.
- Heinze K, Sasaki E, King NP, Baker D, Hilvert D, Wuite GJ, Roos WH. (2016).
Protein Nanocontainers from Nonviral Origin: Testing the Mechanics of Artificial and Natural Protein Cages by AFM.
J Phys Chem B. 07 2016. 120(26):5945-52.
(Abstract)
Self-assembling protein nanocontainers are promising candidates for an increasingly wide scope of purposes. Their applications range from drug delivery vehicles and imaging agents to nanocompartments for controlled enzymatic activity. In order to exploit their full potential in these different fields, characterization of their properties is vital. For example, their mechanical properties give insight into the stability of a particle as a function of their internal content. The mechanics can be probed by atomic force microscopy nanoindentation, and while this single particle method is increasingly used to probe material properties of viral nanocages, it has hardly been used to characterize nonviral nanocages. Here we report nanoindentation studies on two types of nonviral nanocontainers: (i) lumazine synthase from Aquifex aeolicus (AaLS), which naturally self-assembles into icosahedral cages, and (ii) the artificial protein cage O3-33 originating from a computational design approach. In addition, we tested particles that had been engineered toward improved cargo loading capacity and compared these nanocages in empty and loaded states. We found that the thermostable AaLS cages are stiffer and resist higher forces before breaking than the O3-33 particles, but that mutations affecting the size of AaLS particles have a dramatic effect on their structural stability. Furthermore, we show that cargo packaging can occur while maintaining the cage's mechanical properties.
- Bale JB, Gonen S, Liu Y, Sheffler W, Ellis D, Thomas C, Cascio D, Yeates TO, Gonen T, King NP, Baker D. (2016).
Accurate design of megadalton-scale two-component icosahedral protein complexes.
Science. Jul 2016. 353(6297):389-94.
(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.
- Phippen SW, Stevens CA, Vance TD, King NP, Baker D, Davies PL. (2016).
Multivalent Display of Antifreeze Proteins by Fusion to Self-Assembling Protein Cages Enhances Ice-Binding Activities.
Biochemistry. Dec 2016. 55(49):6811-6820.
(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.