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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., Competing Interests: Declaration of interests M.C.M., G.G.H., A.C.W., N.P.K., and D.V. are named as inventors on patent applications filed by the University of Washington related to coronavirus vaccines. The King lab has received unrelated sponsored research agreements from Pfizer and GSK., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Both protein nanoparticle and mRNA vaccines were clinically de-risked during the COVID-19 pandemic 1-6 . These vaccine modalities have complementary strengths: antigen display on protein nanoparticles can enhance the magnitude, quality, and durability of antibody responses 7-10 , while mRNA vaccines can be rapidly manufactured 11 and elicit antigen-specific CD4 and CD8 T cells 12,13 . Here we leverage a computationally designed icosahedral protein nanoparticle that was redesigned for optimal secretion from eukaryotic cells 14 to develop an mRNA-launched nanoparticle vaccine for SARS-CoV-2. The nanoparticle, which displays 60 copies of a stabilized variant of the Wuhan-Hu-1 Spike receptor binding domain (RBD) 15 , formed monodisperse, antigenically intact assemblies upon secretion from transfected cells. An mRNA vaccine encoding the secreted RBD nanoparticle elicited 5- to 28-fold higher levels of neutralizing antibodies than an mRNA vaccine encoding membrane-anchored Spike, induced higher levels of CD8 T cells than the same immunogen when delivered as an adjuvanted protein nanoparticle, and protected mice from vaccine-matched and -mismatched SARS-CoV-2 challenge. Our data establish that delivering protein nanoparticle immunogens via mRNA vaccines can combine the benefits of each modality and, more broadly, highlight the utility of computational protein design in genetic immunization strategies., Competing Interests: Declaration of Interests G.G.H., A.C.W., D.E., J.Y.J.W., M.C.M., D.V., and N.P.K. are named on patents describing designed antigens and nanoparticle immunogens for SARS-CoV-2 and other coronaviruses. The King lab has received unrelated sponsored research agreements from Pfizer and GlaxoSmithKline. N.P. is named on patents describing the use of nucleoside-modified mRNA in lipid nanoparticles as a vaccine platform. N.P. has disclosed those interests fully to the University of Pennsylvania, and he has in place an approved plan for managing any potential conflicts arising from licensing of those patents. N.P. served on the mRNA strategic advisory board of Sanofi Pasteur in 2022 and Pfizer in 2023–2024. N.P. is a member of the Scientific Advisory Board of AldexChem and BioNet, and has consulted for Vaccine Company Inc and Pasture Bio. P.J.C.L., M.M.H.S., and Y.K.T. are employees of Acuitas Therapeutics, a company developing LNP for delivery of mRNA-based therapeutics. Y.K.T. is named on patents describing the use of nucleoside-modified mRNA in lipid nanoparticles as a vaccine platform. The other authors declare no competing interests.

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 2 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 2 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 2 subunits. Immunization of mice with a prefusion-stabilized SARS-CoV-2 S 2 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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests N.P.K. and D.V. are named as inventors on patents for coronavirus nanoparticle vaccines filed by the University of Washington. N.P.K. is a co-founder, shareholder, paid consultant, and chair of the scientific advisory board of Icosavax, Inc. and has received an unrelated sponsored research agreement from Pfizer. D.C. is an employee of Vir Biotechnology and may hold shares in Vir Biotechnology. T.N.S. consults for Apriori Bio on DMS. The lab of T.N.S. has received sponsored research agreements unrelated to the present work from Vir Biotechnology and Aerium Therapeutics, Inc. T.N.S. may receive a share of intellectual property revenue as inventor on a Fred Hutchinson Cancer Center-optioned patent related to stabilization of SARS-CoV-2 RBDs. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Controlling the biodistribution of protein- and nanoparticle-based therapeutic formulations remains challenging. In vivo library selection is an effective method for identifying constructs that exhibit desired distribution behavior; library variants can be selected based on their ability to localize to the tissue or compartment of interest despite complex physiological challenges. Here, we describe further development of an in vivo library selection platform based on self-assembling protein nanoparticles encapsulating their own mRNA genomes (synthetic nucleocapsids or synNCs). We tested two distinct libraries: a low-diversity library composed of synNC surface mutations (45 variants) and a high-diversity library composed of synNCs displaying miniproteins with binder-like properties (6.2 million variants). While we did not identify any variants from the low-diversity surface library that yielded therapeutically relevant changes in biodistribution, the high-diversity miniprotein display library yielded variants that shifted accumulation toward lungs or muscles in just two rounds of in vivo selection. Our approach should contribute to achieving specific tissue homing patterns and identifying targeting ligands for diseases of interest.

As a result of evolutionary selection, the subunits of naturally occurring protein assemblies often fit together with substantial shape complementarity to generate architectures optimal for function in a manner not achievable by current design approaches. We describe a "top-down" reinforcement learning-based design approach that solves this problem using Monte Carlo tree search to sample protein conformers in the context of an overall architecture and specified functional constraints. Cryo-electron microscopy structures of the designed disk-shaped nanopores and ultracompact icosahedra are very close to the computational models. The icosohedra enable very-high-density display of immunogens and signaling molecules, which potentiates vaccine response and angiogenesis induction. Our approach enables the top-down design of complex protein nanomaterials with desired system properties and demonstrates the power of reinforcement learning in protein design.

A survey asked medical social workers and visiting public health nurses to identify health problems and to compare barriers to health care experienced by immigrants and nonimmigrants in the Twin Cities area. Respondents considered infectious diseases a more significant problem for immigrants; they saw alcohol and chemical dependency as more problematic for nonimmigrants. Survey respondents thought that both groups experienced many of the same logistical barriers, including inadequate insurance coverage, transportation, mental stress, time constraints, and distance to their health care facilities. They identified language barriers and the need for trained interpreters as barriers for immigrants only. The survey confirmed widely held assumptions that cultural barriers are more problematic for immigrants, although these barriers were also perceived for nonimmigrants. Participants identified 16 of 18 health services as more available for nonimmigrants. Emergency and obstetrical services are equally available to the two groups, according to survey respondents.