Computational design of self-assembling proteins has long relied on pre-existing structures and sequences, fundamentally limiting control over their structural and functional properties. Recent machine learning-based methods have transformed our ability to design functional small de novo proteins and oligomers, yet methods to design large de novo protein assemblies with structures tailored to specific applications are still underexplored. Here, we develop a generalizable method for designing de novo symmetric protein complexes that incorporate target functional motifs into their structures. We report 34 new protein nanoparticles that form on-target assemblies with cubic point group symmetries. The nanoparticles exhibit a wide variety of backbones that were designed with atom-level accuracy, as evidenced by several cryo-EM and crystal structures that reveal minimal deviations from the design models. We use the method to generate a de novo antigen-tailored nanoparticle vaccine that elicits robust immune responses in mice. These results establish a generalizable approach that can be used to design functional self-assembling protein complexes with structures tailored to specific applications.

Urinary tract infections, caused primarily by uropathogenic E. coli, are a significant public health burden, affecting approximately 50% of women worldwide. The adhesin FimH is responsible for host receptor binding and is therefore a promising vaccine candidate, but prior studies showed that recombinant monomeric FimH is poorly immunogenic. Here we displayed FimH antigens on the two-component protein nanoparticle I53-50 to generate nanoparticle immunogens that elicit robust levels of receptor-blocking antibodies in mice and non-human primates. We produced nanoparticle immunogens displaying either the FimH lectin domain or a recently reported conformationally stabilized antigen, FimH-DSG, comprising both the lectin and pilin domains. When formulated on aluminum hydroxide, both nanoparticle immunogens elicited similar levels of receptor-blocking activity as a ten-fold higher dose of monomeric FimH-DSG formulated with a potent adjuvant. The improved manufacturability of the stabilized antigen, combined with the ability of nanoparticle display to obviate the need for complex adjuvants, provides important preclinical data for FimH-based vaccines intended to prevent urinary tract infections. More broadly, our data extend the applicability of the I53-50 nanoparticle platform, which to date has been mainly used for displaying viral and protozoan antigens, to bacterial indications.

While natural infections expose the immune system for days to weeks of inflammation and antigen presentation, immunizations with conventional bolus vaccines often lead to rapid clearance of antigens and adjuvants. Prolonged exposure to vaccines using controlled delivery devices or repeated dosing regimens has been shown to enhance germinal center reactions, leading to improved humoral responses, including increased magnitude of antibody titers and enhanced neutralizing activity. Herein, we report the use of injectable polymer-nanoparticle (PNP) hydrogels as a vaccine depot technology for sustained delivery of the clinically relevant SARS-CoV-2 Hexapro subunit antigen and a toll-like receptor agonist adjuvant. In mice, we demonstrated that PNP hydrogel vaccines enhanced germinal center responses and antibody responses relative to bolus vaccination. In nonhuman primates, hydrogel vaccines induced enhanced and durable antibody responses against wildtype and variants of concern such as Omicron BA.5 compared to bolus vaccination. We report the first use of a biomaterials-based approach for sustained delivery of vaccines in nonhuman primates, further advancing toward clinical translation.

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. Several transmission-blocking vaccine candidates based on Pfs230 and Pfs48/45 are in clinical development, but it remains unclear whether they will demonstrate high efficacy. Here, the authors develop a stabilized chimeric antigen presenting potent epitopes from Pfs230 and Pfs48/45 in a single construct and demonstrate induction of transmission-reducing antibodies when female mice are immunized with the antigen in a self-assembling protein nanoparticle formulation.

Developing therapies and vaccines against integral membrane proteins is hindered by their extensive hydrophobic surfaces, which complicate production and structural analysis. Here, we describe a general deep learning-based design approach for solubilizing native membrane proteins while preserving their sequence, fold, active-site, and ligand-binding properties. Genetically encoded de novo protein WRAPs [water-soluble RFdiffused amphipathic proteins] surround the lipid-interacting hydrophobic surfaces, rendering them thermostable and water-soluble without the need for detergents. We design WRAPs for both monomeric and oligomeric beta-barrel outer membrane proteins and helical multipass transmembrane proteins. A 2.95-angstrom-resolution cryo-electron microscopy structure of WRAPed mycobacterial porin demonstrates that WRAPs can be used for the structural determination of membrane proteins in solution. As a step toward syphilis vaccine development, we generated soluble versions of Treponema pallidum antigens.

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised neutralizing antibody responses elicited by prior infection or vaccination and abolished the utility of most monoclonal antibody therapeutics. We previously described a computationally-designed, homotrimeric miniprotein inhibitor, designated TRI2-2, that protects mice against pre-Omicron SARS-CoV-2 variants. Here, we show that TRI2-2 exhibits broadly neutralizing activity of SARS-CoV-2 variants and protects mice against BQ.1.1, XBB.1.5 and BA.2.86 challenge when administered intranasally post-exposure. The resistance of TRI2-2 to viral escape by most variants and the ability to deliver it directly to the upper airways highlight the potential of the multivalent miniprotein inhibitor as an alternative therapeutic modality.

Both Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) are members of the genus Ebolavirus and cause outbreaks marked by high fatality rates and repeated spillover from animal reservoirs. Filoviral glycoproteins (GPs) are the primary targets of neutralizing antibodies and form the basis of current vaccines. Here we describe the design, structural characterization, and evaluation of two-component self-assembling icosahedral I53-50 nanoparticles displaying prefusion EBOV or SUDV GP antigens, either individually or in cocktail and mosaic multivalent formats. EBOV-GP-I53-50 and SUDV-GP-I53-50 nanoparticles elicited strong homologous protection in mice and guinea pigs. In the mouse-adapted EBOV model (maEBOV), mosaic and cocktail formulations produced weak survival below that of the matched EBOV-GP-I53-50 GP immunogen. In contrast, in the gpaSUDV guinea pig model (gpSUDV), cocktail and mosaic nanoparticles elicited robust protection against gpSUDV, and detectable antibody responses to both SUDV and EBOV GPs. These findings demonstrate that multivalent GP-I53-50 nanoparticle immunogens can protect rodents from death, severe clinical signs of disease, and weight loss in relevant models. Together with the established clinical safety of the I53-50 platform, these results support continued efforts toward the development of a pan-ebolavirus vaccine.

Underprocessed oligomannose glycans on protein nanoparticle immunogens engage the innate immune system through mannose-binding lectin and complement, enhancing immunogen trafficking and B cell responses. However, the extent to which oligomannose glycans directly improve protective immunity has remained unclear. Here we generate a series of CSP-bearing I53-50 nanoparticle malaria vaccine candidates with defined numbers and types of engineered N-linked glycans and systematically evaluate their immunogenicity and protective efficacy. Oligomannose display enhanced early plasmablast and germinal center B cell responses, leading to increased CSP-specific memory B cells, long-lived plasma cells, and durable serum antibody titers. Furthermore, nanoparticles bearing oligomannose glycans conferred the strongest protection against sporozoite challenge. By comparing immunogens with defined glycoforms, we attribute improved immune responses and protection specifically to oligomannose rather than complex or truncated glycans. These results will help guide the development of general strategies for glycan engineering aimed at enhancing the protective efficacy of nanoparticle vaccines.

Abstract The rapid emergence of SARS-CoV-2 variants that evade neutralizing antibodies underscores the need for next-generation antiviral biologics that combine molecular precision with scalable, cost-effective manufacturing. Computationally designed miniproteins targeting the receptor-binding domain (RBD) of the spike protein offer a compelling alternative to monoclonal antibodies due to their small size, high thermal stability, and compatibility with microbial expression systems. Here we report the end-to-end development and cGMP production of IPD-52520, a de novo antiviral miniprotein, using an optimized E. coli platform. Two miniprotein candidates, a homotrimeric construct (Trimer is referred to as IPD-52520, 17 kDa) and a tandem fusion (Daisy is referred to as IPD-52521, 25 kDa), were evaluated in parallel through systematic optimization of strain selection, media composition, fed-batch fermentation, inclusion-body solubilization, refolding, and chromatographic purification. The Trimer was downselected as the lead molecule based on superior preclinical efficacy, favorable pharmacokinetic properties, and higher volumetric manufacturing yields. The optimized process delivers approximately 2 g/L of purified protein at greater than 90% purity. Scale-up from 5 L to 50 L under cGMP conditions demonstrated excellent batch-to-batch reproducibility across six independent batches, supporting nonclinical and Phase 1 clinical supply. Comprehensive biophysical characterization confirmed a well-folded, predominantly alpha-helical trimer ( T m = 73.4 °C; polydispersity = 1.005) with an intact primary structure and strong target-binding affinity ( K D < 1 pM). Real-time stability studies indicate that the drug substance is stable at 2–8 °C for at least 12 months, with ongoing stability studies. These results demonstrate the feasibility of translating computationally designed antiviral miniproteins into manufacturable biologics and provide a platform applicable to rapid-response therapeutics against current and future pandemic threats.

Both Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) are members of the family Filoviridae, first discovered in 1976 during outbreaks of hemorrhagic fever in northern Zaire and southern Sudan. Ebola virus disease outbreaks are major public health events because of their potential for human-to-human transmission with high case fatality rates. Filoviral surface glycoproteins (GPs) are known to be the primary targets of neutralizing antibodies for protection from disease, and are the relevant immunogens in the two approved EBOV vaccines. Here we describe the design, electron microscopy-based structural characterization, and efficacy testing of a series of icosahedral I53-50 nanoparticles displaying prefusion trimeric EBOV and SUDV GP antigens. Mice and guinea pigs vaccinated with either a cocktail of EBOV-GP-I53-50 plus SUDV-GP-I53-50 or mosaic EBOV / SUDV-GP-I53-50 nanoparticles were protected from death or severe clinical signs of disease and weight loss, respectively, when challenged with either mouse-adapted EBOV or guinea pig-adapted SUDV. ### Competing Interest Statement L.J.S, N.P.K, D.B., L.C., A.J.B., C.W. and N.B. are listed as inventors or major contributors on records of innovation at the University of Washington and an associated provisional patent application that incorporates discoveries described in this manuscript. The King and Baker laboratories have received unrelated sponsored research agreements from Pfizer and Merck respectively. B.F. is an employee of AstraZeneca, Icosavax which is developing I53-50 based nanoparticle vaccines for respiratory syncytial virus and human metapneumovirus. All other authors declare no competing interests.

Vaccines against emerging SARS-CoV-2 variants and sarbecoviruses with pandemic potential must elicit a robust humoral immune response in a population imprinted with the SARS-CoV-2 spike (S) protein. Here, we designed protein nanoparticle (NP) vaccines co-displaying the SARS-CoV-2 BA.5, SARS-CoV-1, and BtKY72 receptor-binding domains (RBDs) with or without the Wuhan-Hu-1 (Wu) RBD. We show that these vaccines elicit cross-reactive and broadly neutralizing plasma antibody responses against SARS-CoV-2 variants and sarbecoviruses in naive and pre-immune animals. Immunization with multivalent RBD-NPs overcomes immune imprinting and elicits neutralizing antibodies and memory B cells specific for the BA.5, SARS-CoV-1, and BtKY72 RBDs in mRNA-1273-vaccinated non-human primates. Multivalent RBD-NPs outperform a monovalent Wu RBD-NP vaccine by providing superior protection in mice and non-human primates challenged with the vaccine-mismatched SARS-CoV-2 XBB.1.5 or the pre-emergent RsSHC014. These data support the use of multivalent RBD-NP vaccines for SARS-CoV-2 variants and sarbecoviruses in naive and pre-immune populations.

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.

Though recombinant protein therapeutics hold great potential in treating many diseases, their intravenous delivery introduces challenges with off-target effects and short circulation half-lives. Injectable biomaterial depots have proven useful in confining therapeutic administration to specific bodily locations but have faced difficulties in simultaneously controlling drug release, network mechanics, and functionalization. Toward addressing these limitations, this work introduces the first recombinant protein-based interpenetrating polymer network (IPN), which we exploit for injectable therapeutic deposition. Each of the self-sorting telechelic biopolymer networks is comprised of an intrinsically disordered XTEN protein midblock differentially flanked with one of two orthogonally self-assembling coil domains that enable rapid shear-thinning and self-healing responsiveness in biomaterials with tunable viscoelasticity. Exploiting the orthogonal and genetically encoded click-like SpyLigation/SnoopLigation chemistries to independently tether proteins-of-interest to each underlying network, we demonstrate that fluorescent proteins and growth factors (rhIGF-1, rhEGF) can be released in a controlled fashion from materials with tunable viscoelasticity while retaining high bioactivity following network dissolution. Such recombinant IPN biomaterials offer exciting opportunities for next-generation biotherapeutic delivery.

Nanoparticles can be coated with targeting ligands to deliver medical agents to specific cells. Serum protein adsorption affects the binding of nanoparticles to target cells. We hypothesized that serum proteins and target receptors compete for binding to nanoparticles. We tested the serum protein binding affinity of 251 nanoparticle designs. Here, we discovered that the binding affinities of serum proteins and receptors to a nanoparticle determine whether it can bind to target cells. We developed and validated a quantitative metric, the binding ratio, to identify nanoparticle designs that can bind to targets in serum with 90% sensitivity and 88% specificity. Using the binding ratio as a numerical guideline for nanoparticle design enabled us to improve the efficiency of nanoparticle binding to target cellular receptors.

The development of therapies and vaccines targeting integral membrane proteins has been complicated by their extensive hydrophobic surfaces, which can make production and structural characterization difficult. Here we describe a general deep learning-based design approach for solubilizing native membrane proteins while preserving their sequence, fold, and function using genetically encoded de novo protein WRAPs (Water-soluble RFdiffused Amphipathic Proteins) that surround the lipid-interacting hydrophobic surfaces, rendering them stable and water-soluble without the need for detergents. We design WRAPs for both beta-barrel outer membrane and helical multi-pass transmembrane proteins, and show that the solubilized proteins retain the binding and enzymatic functions of the native targets with enhanced stability. Syphilis vaccine development has been hindered by difficulties in characterizing and producing the outer membrane protein antigens; we generated soluble versions of four Treponema pallidum outer membrane beta barrels which are potential syphilis vaccine antigens. A 4.0 Å cryo-EM map of WRAPed TP0698 is closely consistent with the design model. WRAPs should be broadly useful for facilitating biochemical and structural characterization of integral membrane proteins, enabling therapeutic discovery by screening against purified soluble targets, and generating antigenically intact immunogens for vaccine development.

Discrete protein assemblies ranging from hundreds of kilodaltons to hundreds of megadaltons in size are a ubiquitous feature of biological systems and perform highly specialized functions1-3. Despite remarkable recent progress in accurately designing new self-assembling proteins, the size and complexity of these assemblies has been limited by a reliance on strict symmetry4,5. Inspired by the pseudosymmetry observed in bacterial microcompartments and viral capsids, we developed a hierarchical computational method for designing large pseudosymmetric self-assembling protein nanomaterials. We computationally designed pseudosymmetric heterooligomeric components and used them to create discrete, cage-like protein assemblies with icosahedral symmetry containing 240, 540, and 960 subunits. At 49, 71, and 96 nm diameter, these nanoparticles are the largest bounded computationally designed protein assemblies generated to date. More broadly, by moving beyond strict symmetry, our work represents an important step towards the accurate design of arbitrary self-assembling nanoscale protein objects.

Interleukin (IL)-23 and IL-17 are well-validated therapeutic targets in autoinflammatory diseases. Antibodies targeting IL-23 and IL-17 have shown clinical efficacy but are limited by high costs, safety risks, lack of sustained efficacy, and poor patient convenience as they require parenteral administration. Here, we present designed miniproteins inhibiting IL-23R and IL-17 with antibody-like, low picomolar affinities at a fraction of the molecular size. The minibinders potently block cell signaling in vitro and are extremely stable, enabling oral administration and low-cost manufacturing. The orally administered IL-23R minibinder shows efficacy better than a clinical anti-IL-23 antibody in mouse colitis and has a favorable pharmacokinetics (PK) and biodistribution profile in rats. This work demonstrates that orally administered de novo-designed minibinders can reach a therapeutic target past the gut epithelial barrier. With high potency, gut stability, and straightforward manufacturability, de novo-designed minibinders are a promising modality for oral biologics.

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.

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised neutralizing antibody responses elicited by prior infection or vaccination and abolished the utility of most monoclonal antibody therapeutics. We previously described a computationally-designed, homotrimeric miniprotein inhibitor, designated TRI2-2, that protects mice against pre-Omicron SARS-CoV-2 variants. Here, we show that TRI2-2 exhibits pan neutralization of variants that evolved during the 4.5 years since the emergence of SARS-CoV-2 and protects mice against BQ.1.1, XBB.1.5 and BA.2.86 challenge when administered post-exposure by an intranasal route. The resistance of TRI2-2 to viral escape and its direct delivery to the upper airways rationalize a path toward clinical advancement.