King Lab | Publications

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2026

• Stabilization of the H5 clade 2.3.4.4b hemagglutinin improves vaccine-elicited neutralizing antibody responses in mice
  Vaccines Antigen Design Influenza MRNA
  Dosey A, Dadonaite B, Gillespie RA, Leaf EM, Vukovich MJ, McGowan J, Grey E, Muramatsu H, Jun RHJ, Pardi N, Kanekiyo M, Bloom JD, King NP.
  (2026) Sci Transl Med. 18(839) :eaea8770 | doi:10.1126/scitranslmed.aea8770 PDF Abstract
  Transmission of highly pathogenic avian influenza from H5 clade 2.3.4.4b has expanded in recent years to infect large populations of birds and mammals, heightening the risk of a human pandemic. Influenza viruses that are adapted to transmission in birds and a variety of mammals tend to have a less stable hemagglutinin (HA) than seasonal influenza viruses, enabling membrane fusion at comparatively higher pH levels. Here, we combined five mutations in the H5 HA that increased its melting temperature and promoted stable closure of the HA trimer. Structural analysis by cryo-electron microscopy revealed that the stabilizing mutations create several new hydrophobic interactions while maintaining the local HA structure. We found that vaccinating mice with stabilized H5 HA immunogens resulted in higher hemagglutination inhibition and neutralization titers than nonstabilized comparators. Epitope mapping of vaccine-elicited polyclonal antibody responses using negative-stain electron microscopy and deep mutational scanning showed that site E on the side of the HA receptor binding domain was immunodominant across all groups; however, the stabilized immunogens shifted responses toward the receptor binding site, which elicited a higher proportion of neutralizing antibodies. Consistent with these findings, stabilized H5 HA immunogens delivered as messenger RNA-lipid nanoparticle (mRNA-LNP) vaccines protected mice against H5N1 challenge. These findings highlight that H5 HA-stabilizing mutations enhance the quality of antibody responses across different vaccine formats, underscoring their potential to improve pandemic preparedness vaccines targeting viruses from this widely circulating clade.
• A stabilized tandem antigen chimera that elicits potent malaria transmission-reducing activity
  Vaccines Antigen Design Nanoparticles Malaria
  Ivanochko D, Miura K, Hailemariam S, Ravichandran R, Song Y, Huang WC, Stoter R, Teelen K, van Gemert GJ, Leaf EM, Chan S, Men C, Semesi A, Shiu C, MacGill RS, Long CA, Jore MM, King NP, Lovell JF, Julien JP.
  (2026) Nat Commun. | doi:10.1038/s41467-026-68761-1 PDF Abstract
  Malaria parasite transmission remains a barrier to elimination since asymptomatic individuals sustain the infectious reservoir. Transmission-blocking vaccine (TBV) candidates targeting Plasmodium falciparum (Pf) gametocyte surface proteins Pfs230 and Pfs48/45 have shown promise in clinical trials. Several vaccine candidates have been developed for these antigens, yet it is unclear which elicit the most robust and durable transmission-blocking responses. From structure-function relationships of monoclonal antibodies in complex with both antigens, we report the development of a stabilized tandem antigen chimera (STAC), which presents the most potent epitopes from Pfs230 domain 1 (Pfs230-D1) and Pfs48/45 domain 3 (Pfs48/45-D3) in a single construct, while masking non-functional epitopes using an engineered pseudo-native domain disposition. Iterative structure-guided optimization improved antigen yields and stability, while nanoparticle-based multimerization enhanced the functional transmission-reducing activity elicited by the immunogen in female mice. Immunizations with STAC genetically conjugated to self-assembling protein nanoparticles elicited antibodies with potent transmission-reducing activity comparable or superior to the multimerized Pfs230-D1 and Pfs48/45-D3. These findings establish STAC as a promising next-generation TBV candidate to disrupt malaria transmission and accelerate elimination efforts. More broadly, our results support the engineering of highly ordered and stable multi-domain antigens in a single protein as a strategy for the cost-efficient development of multi-component vaccines.

2025

• Computationally designed mRNA-launched protein nanoparticle immunogens elicit protective antibody and T cell responses in mice
  Vaccines MRNA Nanoparticles CoV Lab-led
  Hendricks GG, Grigoryan L, Navarro MJ, Catanzaro NJ, Hubbard ML, Powers JM, Mattocks M, Treichel C, Walls AC, Lee J, Ellis D, Wang JYJ, Cheng S, Miranda MC, Valdez A, Chao CW, Chan S, Men C, Johnson MR, Zepeda SK, Ols S, Hui H, Wu SY, Lujan V, Muramatsu H, Lin PJC, Sung MMH, Tam YK, Leaf EM, Pardi N, Baric RS, Pulendran B, Veesler D, Schäfer A, King NP.
  (2025) Sci Transl Med. 17(820) :eadu2085 | doi:10.1126/scitranslmed.adu2085 PDF Abstract
  Messenger RNA (mRNA) vaccines and computationally designed protein nanoparticle vaccines were both clinically derisked and licensed for the first time during the coronavirus disease 2019 (COVID-19) pandemic. These vaccine modalities have complementary immunological benefits that provide strong motivation for their combination. Here, we demonstrate proof of concept for genetic delivery of computationally designed protein nanoparticle immunogens. Using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a model system, we genetically fused a stabilized variant of the Wuhan-Hu-1 spike protein receptor binding domain (RBD) to a protein nanoparticle we previously designed for optimal secretion from human cells. Upon secretion, the nanoparticle formed monodisperse and antigenically intact assemblies displaying 60 copies of the RBD in an immunogenic array. Compared with mRNA vaccines encoding membrane-anchored spike protein and a secreted RBD trimer, an mRNA vaccine encoding the RBD nanoparticle elicited 5- to 28-fold higher titers of neutralizing antibodies in mice. In addition, the “mRNA-launched” RBD nanoparticle vaccine induced higher frequencies of antigen-specific CD8 T cells than the same immunogen delivered as adjuvanted protein and protected mice from either Wuhan-Hu-1 or Omicron BA.5 challenge. These results establish that delivering computationally designed protein nanoparticle immunogens through mRNA can combine the benefits of both vaccine modalities. More broadly, our data highlight the utility of computational protein design in genetic vaccination strategies.
• Safety and immunogenicity of a homologous booster dose of a SARS-CoV-2 recombinant protein nanoparticle vaccine (GBP510) adjuvanted with AS03: 12 months follow-up result of an open-label, non-randomised extension of a phase 1/2 trial
  Vaccines Nanoparticles CoV Clinical Trials
  Song JY, Choi WS, Heo JY, Kim YR, Lee JS, Jung DS, Kim SW, Park KH, Eom JS, Jeong SJ, Lee J, Kwon KT, Choi HJ, Sohn JW, Kim YK, Lee Y, Park HK, Ryu JH, Lee SJ, Park YW, Kim H, Solmi F, Ceregido MA, Koutsoukos M, King N, Veesler D, Cheong HJ.
  (2025) Int J Infect Dis. :108108 | doi:10.1016/j.ijid.2025.108108 PDF Abstract
  This study evaluated the safety and immunogenicity of a homologous GBP510/AS03 booster given 6-12 months after a primary two-dose series.
• Elicitation of liver-stage immunity by nanoparticle immunogens displaying P. falciparum CSP-derived antigens
  Vaccines Nanoparticles Lab-led
  Langowski MD, Francica JR, Roederer AL, Hurlburt NK, Rodarte JV, Da Silva Pereira L, Flynn BJ, Bonilla B, Dillon M, Kiyuka P, Ravichandran R, Weidle C, Carter L, Rao M, Matyas GR, Pepper M, Idris AH, Seder RA, Pancera M, King NP.
  (2025) NPJ Vaccines. 10(1) :87 | doi:10.1038/s41541-025-01140-x PDF Abstract
  A vaccine that provides robust, durable protection against malaria remains a global health priority. Although a breakthrough in the fight against malaria has recently been achieved by the licensure of two vaccines based on the circumsporozoite protein (CSP), the effectiveness and durability of protection can still be improved. Both vaccines contain a portion of CSP that does not include epitopes targeted by recently identified, potently protective monoclonal antibodies, suggesting that newer immunogens can expand the breadth of immunity and potentially increase protection. Here we explored >100 alternative CSP-based immunogens and evaluated the immunogenicity and protection of a large number of candidates, comparing several to the licensed R21 vaccine. The data highlight several general features that improve the stability and immunogenicity of CSP-based vaccines, such as inclusion of the C-terminal domain and high-density display on protein nanoparticle scaffolds. We also identify antigen design strategies that do not warrant further exploration, such as synthetic repeat regions that include non-native repeat cadences. The benchmark R21 vaccine outperformed our best immunogen for immunogenicity and protection. Overall, our data provide valuable insights on the inclusion of junctional region epitopes that will guide the development of potent and durable vaccines against malaria.

2024

• Protein nanoparticle vaccines induce potent neutralizing antibody responses against MERS-CoV
  Vaccines Nanoparticles Lab-led CoV
  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.
• Potent neutralization of SARS-CoV-2 variants by RBD nanoparticle and prefusion-stabilized spike immunogens
  Vaccines Nanoparticles CoV 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 PDF 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
  Vaccines Nanoparticles HIV
  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 PDF 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.
• A broadly generalizable stabilization strategy for sarbecovirus fusion machinery vaccines
  Vaccines Antigen Design CoV
  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 PDF 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
  Vaccines MRNA Nanoparticles Herpesviruses
  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 PDF 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.

2023

• Combinatorial immune refocusing within the influenza hemagglutinin RBD improves cross-neutralizing antibody responses
  Vaccines Nanoparticles Antigen Design Influenza 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 Antigen Design
  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
  Vaccines CoV
  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 PDF 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.
• Nanoparticle display of prefusion coronavirus spike elicits S1-focused cross-reactive antibody response against diverse coronavirus subgenera
  Vaccines Nanoparticles CoV
  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 PDF 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
  Vaccines Nanoparticles Antibodies RSV Lab-led
  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 PDF 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.
• Broadly neutralizing antibodies against sarbecoviruses generated by immunization of macaques with an AS03-adjuvanted COVID-19 vaccine
  Vaccines Nanoparticles Antibodies CoV
  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 PDF 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.
• Progress in vaccine development for infectious diseases-a Keystone Symposia report
  Vaccines
  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 PDF 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
  Vaccines Translation Nanoparticles CoV
  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.
  (2023) Process Biochem. 129:241-256 | doi:10.1016/j.procbio.2023.03.014 PDF 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.
• Design and immunological evaluation of two-component protein nanoparticle vaccines for East Coast fever
  Vaccines Nanoparticles Livestock 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.
  (2022) Front Immunol. 13:1015840 | doi:10.3389/fimmu.2022.1015840 PDF 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.

2022

• Induction of cross-neutralizing antibodies by a permuted hepatitis C virus glycoprotein nanoparticle vaccine candidate
  Vaccines Nanoparticles Antigen Design HCV
  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 PDF 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
  Vaccines Nanoparticles CoV
  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 PDF 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 PDF 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.
• Durable protection against the SARS-CoV-2 Omicron variant is induced by an adjuvanted subunit vaccine
  Vaccines Nanoparticles CoV
  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 PDF 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
  Vaccines Nanoparticles Antigen Design Malaria
  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 PDF 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
  Vaccines Nanoparticles CoV
  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 PDF 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.
• 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
  Vaccines Nanoparticles Clinical Trials CoV
  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.
  (2022) EClinicalMedicine. 51:101569 | doi:10.1016/j.eclinm.2022.101569 PDF 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
  Vaccines Nanoparticles Herpesviruses
  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 PDF 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.
• Epitope-focused immunogen design based on the ebolavirus glycoprotein HR2-MPER region
  Vaccines Antigen Design Nanoparticles Ebola
  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 PDF 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.
• Structure-based design of stabilized recombinant influenza neuraminidase tetramers
  Vaccines Antigen Design Influenza 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 PDF 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.

2021

• Limited access to antigen drives generation of early B cell memory while restraining the plasmablast response
  Vaccines Antibodies
  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 PDF 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
  Vaccines Antibodies Malaria
  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) J Exp Med. 218(4) | doi:10.1084/jem.20200942 PDF 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.
• Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice
  Vaccines Antigen Design CoV Nanoparticles
  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.
  (2021) Proc Natl Acad Sci U S A. 118(38) | doi:10.1073/pnas.2106845118 PDF 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 CoV 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 PDF 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.
• Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM
  Vaccines Antibodies Nanoparticles HIV
  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 PDF 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 CoV 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.
  (2021) Front Immunol. 12:710263 | doi:10.3389/fimmu.2021.710263 PDF 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.
• Quadrivalent influenza nanoparticle vaccines induce broad protection
  Vaccines Nanoparticles Influenza 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 PDF 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.
• Immunofocusing and enhancing autologous Tier-2 HIV-1 neutralization by displaying Env trimers on two-component protein nanoparticles
  Vaccines Nanoparticles HIV Lab-led
  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 PDF 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 Nanoparticles CoV
  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 PDF 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

• Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2
  Vaccines Nanoparticles CoV Lab-led
  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 PDF 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.
• A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver
  Vaccines Antibodies Malaria
  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 PDF 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.
• Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens
  Vaccines Nanoparticles HIV
  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 PDF 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.
• Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations
  Vaccines Nanoparticles Glycans HIV
  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 PDF 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 Methods HIV Lab-led
  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) Elife. 9 | doi:10.7554/eLife.57659 PDF 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.
• New Vaccine Design and Delivery Technologies
  Vaccines Review
  Kanekiyo M, Ellis D, King NP.
  (2019) J Infect Dis. 219(Suppl_1) :S88-S96 | doi:10.1093/infdis/jiy745 PDF 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

• Enhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle
  Vaccines Nanoparticles HIV Lab-led
  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 PDF 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.
• Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus
  Vaccines Nanoparticles RSV 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 PDF 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.