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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.

Undergraduate research experiences can improve student success in graduate education and STEM careers. During the COVID-19 pandemic, undergraduate researchers at our institution and many others lost their work-study research positions due to interruption of in-person research activities. This imposed a financial burden on the students and eliminated an important learning opportunity. To address these challenges, we created a paid, fully-remote, cohort-based research curriculum in computational protein design. Our curriculum used existing protein design methods as a platform to first educate and train undergraduate students and then to test research hypotheses. In the first phase, students learned computational methods to assess the stability of designed protein assemblies. In the second phase, students used a larger dataset to identify factors that could improve the accuracy of current protein design algorithms. This cohort-based program created valuable new research opportunities for undergraduates at our institute and enhanced the undergraduates’ feeling of connection with the lab. Students learned transferable and useful skills such as literature review, programming basics, data analysis, hypothesis testing, and scientific communication. Our program provides a model of structured computational research training opportunities for undergraduate researchers in any field for organizations looking to expand educational access.Graphical Abstract

SUMMARYThe head domain of influenza hemagglutinin (HA) elicits potently neutralizing yet mostly strain-specific antibodies during infection and vaccination. Here we evaluated a series of immunogens that combined several immunofocusing techniques for their ability to enhance the functional breadth of vaccine-elicited immune responses. We designed a series of “trihead” nanoparticle immunogens that display native-like closed trimeric heads from the HAs of several H1N1 influenza viruses, including hyperglycosylated variants and hypervariable variants that incorporate natural and designed sequence diversity at key positions in the periphery of the receptor binding site (RBS). Nanoparticle immunogens displaying triheads or hyperglycosylated triheads elicited higher HAI and neutralizing activity against vaccine-matched and -mismatched H1 viruses than corresponding immunogens lacking either trimer-stabilizing mutations or hyperglycosylation, indicating that both of these engineering strategies contributed to improved immunogenicity. By contrast, mosaic nanoparticle display and antigen hypervariation did not significantly alter the magnitude or breadth of vaccine-elicited antibodies. Serum competition assays and electron microscopy polyclonal epitope mapping revealed that the trihead immunogens, especially when hyperglycosylated, elicited a high proportion of antibodies targeting the RBS, as well as cross-reactive antibodies targeting a conserved epitope on the side of the head. Our results yield important insights into antibody responses against the HA head and the ability of several structure-based immunofocusing techniques to influence vaccine-elicited antibody responses.HIGHLIGHTSGeneralization of trihead antigen platform to several H1 hemagglutinins, including hyperglycosylated and hypervariable variantsTrimer-stabilizing mutations in trihead nanoparticle immunogens lead to lower levels of non-neutralizing antibody responses in both mice and rabbitsHyperglycosylated triheads elicit higher antibody responses against broadly neutralizing epitopes

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and important for targeted delivery of biologics. We describe the design of octahedral non-porous nanoparticles with the three symmetry axes (four-fold, three-fold, and two-fold) occupied by three distinct protein homooligomers: ade novodesigned tetramer, an antibody of interest, and a designed trimer programmed to disassemble below a tunable pH transition point. The nanoparticles assemble cooperatively from independently purified components, and a cryo-EM density map reveals that the structure is very close to the computational design model. The designed nanoparticles can package a variety of molecular payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between to 5.9-6.7. To our knowledge, these are the first designed nanoparticles with more than two structural components and with finely tunable environmental sensitivity, and they provide new routes to antibody-directed targeted delivery.

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.

Most vaccines require several immunizations to induce robust immunity, and indeed, most SARS-CoV-2 vaccines require an initial two-shot regimen followed by several boosters to maintain efficacy. Such a complex series of immunizations unfortunately increases the cost and complexity of populations-scale vaccination and reduces overall compliance and vaccination rate. In a rapidly evolving pandemic affected by the spread of immune-escaping variants, there is an urgent need to develop vaccines capable of providing robust and durable immunity. In this work, we developed a single immunization SARS-CoV-2 subunit vaccine that could rapidly generate potent, broad, and durable humoral immunity. We leveraged injectable polymer-nanoparticle (PNP) hydrogels as a depot technology for the sustained delivery of a nanoparticle COVID antigen displaying multiple copies of the SARS-CoV-2 receptor-binding-domain (RBD-NP), and potent adjuvants including CpG and 3M-052. Compared to a clinically relevant prime-boost regimen with soluble vaccines formulated with CpG/Alum or 3M-052/Alum adjuvants, PNP hydrogel vaccines more rapidly generated higher, broader, and more durable antibody responses. Additionally, these single-immunization hydrogel-based vaccines elicited potent and consistent neutralizing responses. Overall, we show that PNP hydrogels elicit improved anti-COVID immune responses with only a single administration, demonstrating their potential as critical technologies to enhance our overall pandemic readiness.

Computationally designed multi-subunit assemblies have shown considerable promise for a variety of applications, including a new generation of potent vaccines. One of the major routes to such materials is rigid body sequence-independent docking of cyclic oligomers into architectures with point group or lattice symmetries. Current methods for docking and designing such assemblies are tailored to specific classes of symmetry and are difficult to modify for novel applications. Here we describe RPXDock, a fast, flexible, and modular software package for sequence-independent rigid-body protein docking across a wide range of symmetric architectures that is easily customizable for further development. RPXDock uses an efficient hierarchical search and a residue-pair transform (RPX) scoring method to rapidly search through multidimensional docking space. We describe the structure of the software, provide practical guidelines for its use, and describe the available functionalities including a variety of score functions and filtering tools that can be used to guide and refine docking results towards desired configurations.Author SummaryProtein design methodologies are now able to generate, through a stepwise approach, a wide variety of self-assembling protein structures that begin to rival the structural complexity of naturally occurring protein nanomachines. Efficient methods for docking oligomeric protein building blocks in user-defined target symmetries are central to these techniques. We developed RPXDock as a fast and versatile method to systematically dock pre-existing proteins together into a multitude of asymmetrical and symmetrical architectures. RPXdock is also readily extendable to future applications through the addition of new symmetries, score functions, and filtering criteria.

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.

The multisubunit protein assemblies that play critical roles in biology are the result of evolutionary selection for function of the entire assembly, and hence the subunits in structures such as icosahedral viral capsids often fit together with remarkable shape complementarity1,2. In contrast, the large multisubunit assemblies that have been created by de novo protein design, notably the icosahedral nanocages used in a new generation of potent vaccines3–7, have been built by first designing symmetric oligomers with cyclic symmetry and then assembling these into nanocages while keeping the internal structure fixed8–14, which results in more porous structures with less extensive shape matching between the components. Such hierarchical “bottom-up” design approaches have the advantage that one interface can be designed and validated in the context of the cyclic oligomer building block15,16, but the disadvantage that the structural and functional features of the assemblies are limited by the properties of the predesigned building blocks. To overcome this limitation, we set out to develop a “top-down” reinforcement learning based approach to protein nanomaterial design in which both the structures of the subunits and the interactions between them are built up coordinately in the context of the entire assembly. We developed a Monte Carlo tree search (MCTS) method17,18 which assembles protein monomer structures in the context of an overall architecture guided by a loss function which enables specification of any desired overall structural properties such as shape and porosity. We demonstrate the power of the approach by designing hyperstable icosahedral assemblies more compact than any previously observed protein icosahedral structure (designed or naturally occurring), that have very low porosity and are robust to fusion and display of proteins as complex as influenza hemagglutinin. CryoEM structures of two designs are very close to the computational design models. Our top-down reinforcement learning approach should enable the design of a wide variety of complex protein nanomaterials by direct optimization of overall system properties.

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.

Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. Retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the novel nanoparticles we describe may be broadly useful in biotechnological applications.

Humoral immunity to SARS-CoV-2 can be supplemented with polyclonal sera from convalescent donors or an engineered monoclonal antibody (mAb) product. While pentameric IgM antibodies are responsible for much of convalescent sera’s neutralizing capacity, all available mAbs are based on the monomeric IgG antibody subtype. We now show that IgM mAbs derived from immune memory B cell receptors are potent neutralizers of SARS-CoV-2. IgM mAbs outperformed clonally identical IgG antibodies across a range of affinities and SARS-CoV-2 receptor-binding domain epitopes. Strikingly, efficacy against SARS-CoV-2 viral variants was retained for IgM but not for clonally identical IgG. To investigate the biological role for IgM memory in SARS-CoV-2, we also generated IgM mAbs from antigen-experienced IgM+ memory B cells in convalescent donors, identifying a potent neutralizing antibody. Our results highlight the therapeutic potential of IgM mAbs and inform our understanding of the role for IgM memory against a rapidly mutating pathogen.

The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).

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 immunization1. Here we show that computationally designed, two-component nanoparticle immunogens2 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 vaccines3. A nanoparticle influenza vaccine candidate is shown to induce broad cross-reactive antibody responses in animal models.

All-trans-retinoic acid (atRA) is a critical endogenous signaling molecule. atRA is predominantly synthesized from retinaldehyde by aldehyde dehydrogenase 1A1 (ALDH1A1), but aldehyde oxidase (AOX) may also contribute to atRA biosynthesis. The goal of this study was to test the hypothesis that AOX contributes significantly to atRA formation in human liver. Human recombinant AOX formed atRA from retinaldehyde (Km ∼1.5 ± 0.4 µM; kcat ∼3.6 ± 2.0 minute-1). In human liver S9 fractions (HLS9), atRA formation was observed in the absence of NAD+, suggesting AOX contribution to atRA formation. In the presence of NAD+, Eadie-Hofstee plots of atRA formation in HLS9 indicated that two enzymes contributed to atRA formation. The two enzymes were identified as AOX and ALDH1A1 based on inhibition of atRA formation by AOX inhibitor hydralazine (20%-50% inhibition) and ALDH1A1 inhibitor WIN18,446 (50%-80%inhibition). The expression of AOX in HLS9 was 9.4-24 pmol mg-1 S9 protein, whereas ALDH1A1 expression was 156-285 pmol mg-1 S9 protein measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of signature peptides. The formation velocity of atRA in the presence of NAD+ correlated significantly with the expression of ALDH1A1 and AOX protein. Taken together, the data show that both AOX and ALDH1A1 contribute to atRA biosynthesis in the human liver, with ALDH1A1 being the high-affinity, low-capacity enzyme and AOX being the low-affinity, high-capacity enzyme. The results suggest that in the case of ALDH1A dysfunction or excess vitamin A, AOX may play an important role in regulating hepatic vitamin A homeostasis and that inhibition of AOX may alter atRA biosynthesis and signaling. SIGNIFICANCE STATEMENT: This study provides direct evidence to show that human AOX converts retinaldehyde to atRA and contributes to hepatic atRA biosynthesis. The finding that AOX may be responsible for 20%-50% of overall hepatic atRA formation suggests that alterations in AOX activity via drug-drug interactions, genetic polymorphisms, or disease states may impact hepatic atRA concentrations and signaling and alter vitamin A homeostasis.

Despite remarkable advances over the past several decades, many therapeutic nanomaterials fail to overcome major in vivo delivery barriers. Controlling immunogenicity, optimizing biodistribution, and engineering environmental responsiveness are key outstanding delivery problems for most nanotherapeutics. However, notable exceptions exist including some lipid and polymeric nanoparticles, some virus-based nanoparticles, and nanoparticle vaccines where immunogenicity is desired. Self-assembling protein nanoparticles offer a powerful blend of modularity and precise designability to the field, and have the potential to solve many of the major barriers to delivery. In this review, we provide a brief overview of key designable features of protein nanoparticles and their implications for therapeutic delivery applications. We anticipate that protein nanoparticles will rapidly grow in their prevalence and impact as clinically relevant delivery platforms.

SummaryDespite the remarkable efficacy of COVID-19 vaccines, waning immunity, and the emergence of SARS-CoV-2 variants such as Omicron represents a major global health challenge. Here we present data from a study in non-human primates demonstrating durable protection against the Omicron BA.1 variant induced by a subunit SARS-CoV-2 vaccine, consisting of RBD (receptor binding domain) on the I53-50 nanoparticle, adjuvanted with AS03, currently in Phase 3 clinical trial (NCT05007951). Vaccination induced robust neutralizing antibody (nAb) titers that were maintained at high levels for at least one year after two doses (Pseudovirus nAb GMT: 2207, Live-virus nAb GMT: 1964) against the ancestral strain, but not against Omicron. However, a booster dose at 6-12 months with RBD-Wu or RBD-β (RBD from the Beta variant) displayed on I53-50 elicited equivalent and remarkably high neutralizing titers against the ancestral as well as the Omicron variant. Furthermore, there were substantial and persistent memory T and B cell responses reactive to Beta and Omicron variants. Importantly, vaccination resulted in protection against Omicron infection in the lung (no detectable virus in any animal) and profound suppression of viral burden in the nares (median peak viral load of 7567 as opposed to 1.3×107 copies in unvaccinated animals) at 6 weeks post final booster. Even at 6 months post vaccination, there was significant protection in the lung (with 7 out of 11 animals showing no viral load, 3 out of 11 animals showing ~20-fold lower viral load than unvaccinated controls) and rapid control of virus in the nares. These results highlight the durable cross-protective immunity elicited by the AS03-adjuvanted RBD-I53-50 nanoparticle vaccine platform.

Summary/AbstractEpstein-Barr virus (EBV) is a cancer-associated pathogen responsible for 140,000 deaths per year. EBV is also the etiological agent of infectious mononucleosis and is associated with multiple sclerosis and rheumatoid arthritis. Thus, an EBV vaccine could alleviate significant morbidity and mortality. EBV is orally transmitted and has tropism for both epithelial cells and B cells which are present in the oral cavity. Therefore, a prophylactic vaccine would need to prevent infection of both cell types. Passive transfer neutralizing monoclonal antibodies targeting the viral 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 produced and evaluated the immunogenicity of several nanoparticle immunogens displaying gH/gL with distinct valencies and geometries. After one or two immunizations, all nanoparticles elicited superior binding and neutralizing titers relative to monomeric gH/gL. Antibodies elicited by a computationally designed self-assembling nanoparticle that displays 60 copies of the gH/gL protein conferred protection against a lethal dose of EBV in a humanized mouse challenge model, whereas antibodies elicited by monomeric gH/gL did not. Taken together, these data motivate further development of gH/gL nanoparticle vaccine candidates for EBV.

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 (VOC) 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 VOC. We report that vaccinated BALB/c mice do not recapitulate faithfully the breadth and potency of neutralizing antibody responses against VOC, as compared to non-human primates or humans, suggesting caution should be exercised when interpreting data for this animal model.

Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor critical for the innate immune response to intracellular pathogens, DNA damage, tumorigenesis and senescence. Binding to double-stranded DNA (dsDNA) induces conformational changes in cGAS that activate the enzyme to produce 2'-3' cyclic GMP-AMP (cGAMP), a second messenger that initiates a potent interferon (IFN) response through its receptor, STING. Here, we combined two-state computational design with informatics-guided design to create constitutively active, dsDNA ligand-independent cGAS (CA-cGAS). We identified CA-cGAS mutants with IFN-stimulating activity approaching that of dsDNA-stimulated wild-type cGAS. DNA-independent adoption of the active conformation was directly confirmed by X-ray crystallography. In vivo expression of CA-cGAS in tumor cells resulted in STING-dependent tumor regression, demonstrating that the designed proteins have therapeutically relevant biological activity. Our work provides a general framework for stabilizing active conformations of enzymes and provides CA-cGAS variants that could be useful as genetically encoded adjuvants and tools for understanding inflammatory diseases.

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., Here, the authors present cryoEMPEM, a method for high-resolution structural analysis of vaccine-elicited polyclonal antibody responses. They apply cryoEMPEM in combination with standard serology experiments to characterize the polyclonal antibody (pAb) responses elicited in rhesus macaques by HIV Env trimer immunogens and were able to determine up to 8 different polyclonal antibody structures in complex with their respective antigen from a single cryoEM dataset.