Germline targeting vaccination strategies against highly variable pathogens such as HIV aim to elicit broadly neutralizing antibodies (bnAbs) with particular immunogenetic or structural features. The V2 apex of the HIV Env protein is a promising target for a class of bnAbs that contain conserved structural motifs in the heavy chain complementarity determining region 3 (CDRH3). Here, we show that these structural motifs are targetable by vaccination by characterizing V2 apex "axe-like" CDRH3s in the human repertoire and developing immunogens capable of engaging them. We determined the frequency and diversity of axe-like CDRH3s in healthy human donors using a series of structural informatics approaches, finding these precursors in nearly 90% of donors. Axe-targeting immunogens based on the HIV Env Q23.17 bound axe-like precursors in cryo-electron microscopy structures, induced V2 apex-specific antibody responses in humanized mice, and induced axe-like heterologous neutralizing antibodies in rhesus macaques infected with a germline-targeted simian-HIV. These results illustrate a structure-guided immunoinformatic vaccine design paradigm that can be employed to elicit immunogenetically diverse yet structurally conserved classes of antibodies.

Current influenza vaccines induce mostly strain-specific immunity necessitating annual reformulation and dosing. Here, we developed an improved seasonal influenza vaccine based on A/H1N1/Wisconsin/588/2019. We designed a DNA-launched self-assembling nanoparticle that displayed seven Wisconsin/588/2019 hemagglutinin (HA) head domains (WI19-7mer). WI19-7mer nanovaccine improved heterologous HAI titers and CD8+ cellular responses in mice than DNA encoded HA trimer (WI19 HA). In human antibody repertoire mice, WI19-7mer induced superior breadth to a diverse panel of H1 HAs compared to WI19 HA immunized animals. Cross-reactive HAI titers were maintained better in mice immunized with WI19-7mer than WI19 HA. The WI19-7mer induced improved antibody binding breadth and provided superior protection in a heterologous challenge compared to challenge-matched HA trimer. Addition of the cytokine adjuvant (CTACK) to WI19-7mer significantly improved breadth, HAI, peripheral responses, and protection in heterologous challenge. These data demonstrate that combining nucleic acid delivery, immune focusing, low valency nanoparticle, and mucosal adjuvant for enhanced vaccine effectiveness has broader applications for other viruses.

Currently approved vaccines for the prevention of malaria provide only partial protection against disease due to high variability in the quality of induced antibodies. These vaccines present the unstructured central repeat region, as well as the C-terminal domain, of the circumsporozoite protein ( Pf CSP) of the malaria parasite, Plasmodium falciparum [K. L. Williams et al ., Nat. Med. 30 ,1–13 (2024)]. A recently discovered protective monoclonal antibody, L9, recognizes three structured copies of the Pf CSP minor repeat. Similarly to other highly potent antimalarial antibodies, L9 relies on critical homotypic interactions between antibodies for its high protective efficacy [P. Tripathi et al. , Structure 31 , 480–491.e4 (2023); G. M. Martin et al. , Nat. Commun. 14 ,2815 (2023)]. Here, we report the design of antigens scaffolding one copy of Pf CSP’s minor repeat capable of binding L9. To design antigens capable of presenting multiple, structure-based epitopes in one scaffold, we developed a machine learning– driven structural antigen design pipeline, MESODID, tailored to focus on multiepitope vaccine targets. We use this pipeline to design multiple scaffolds that present three copies of the Pf CSP minor repeat. A 3.6 Å cryo-EM structure of our top design, minor repeat targeting immunogen (M-TIM), demonstrates that M-TIM successfully orients three copies of L9, effectively recapitulating its critical homotypic interactions. The wide prevalence of repeated epitopes in key vaccine targets, such as HIV-1 Envelope, SARS-CoV-2 spike, and Influenza Hemagglutinin, suggests that MESODID will have broad utility in creating antigens that incorporate such epitopes, offering a powerful approach to developing vaccines against a range of challenging infections, including malaria.

Sequential immunization is a promising approach to elicit broadly neutralizing antibodies (bNAbs) against the HIV-1 Envelope (Env). However, available protocols are inefficient and involve multiple immunizations over long periods of time. Here, we present WIN332, a new engineered Env-immunogen that induces a new class of neutralizing N332-glycan-independent antibodies to the conserved V3-glycan epitope of Env after a single bolus immunization in nonhuman primates. WIN332 binds to precursors of canonical human N332-glycan-dependent (Type-I) V3-glycan bNAbs but also of a first-of-its-class N332-glycan-independent (Type-II) V3-glycan bNAb. A single immunization elicits neutralizing serum and monoclonal antibodies that are boosted and affinity matured with a heterologous immunogen. EMPEM analysis of serum antibodies, antibody cloning and cryo-EM analysis reveal that WIN332 elicits N332-glycan-independent antibodies with remarkable sequence and binding similarities with the most potent human type-I and type-II V3-glycan bNAbs. Thus, WIN332 is a promising vaccine candidate to streamline V3-glycan bNAb elicitation.

Oncogenic forms of HPV account for 4.5% of the global cancer burden worldwide. This includes cervical, vaginal, vulvar, penile, and anal cancers, as well as head and neck cancers. As such, there is an urgent need to develop effective therapeutic vaccines to drive the immune system’s cellular response against cancer cells. One of the primary goals of cancer vaccination is to increase the potency and diversity of anti-tumor T-cell responses; one strategy to do so involves the delivery of full-length cancer antigens scaffolded onto DNA-launched nanoparticles to improve T-cell priming. We developed a platform, making use of structural prediction algorithms such as AlphaFold2, to design stabilized, more full-length antigens of relevant HPV proteins and then display them on nanoparticles. We demonstrated that many such designs for both the HPV16 E6 and E7 antigens assembled and drove strong CD8+ T-cell responses in mice. We further tested nanoparticles in a genetically diverse, more translationally relevant CD-1 mouse model and demonstrated that both E6 and E7 nanoparticle designs drove a CD8+ biased T-cell response. These findings serve as a proof-of-concept study for nanoparticle antigen design as well as identify new vaccine candidates for HPV-associated cancers.

Broadly neutralizing antibodies targeting the V2 apex of HIV-1 envelope are desired as vaccine design templates, but few have been described. Here, we report 11 lineages of V2 apex-neutralizing antibodies from simian-human immunodeficiency virus (SHIV)-infected rhesus macaques and determine cryo-EM structures for 9. A single V2 apex-neutralizing lineage accounted for cross-clade breadth in most macaques, and somatic hypermutation relative to breadth was generally low, exemplified by antibody V033-a.01 with <5% nucleotide mutation and 37% breadth (208-strain panel). Envelope complex structures revealed eight different antibody classes (one multi-donor) and the complete repertoire of all five possible recognition topologies, recapitulating canonical human modes of apex insertion and C-strand hydrogen bonding. Despite this diversity in recognition, all rhesus-V2 apex antibodies were derived from reading frame two of the DH3-15*01 gene. Collectively, these results define-in rhesus-the structural and genetic basis of HIV-1 V2 apex recognition and demonstrate unprecedented structural plasticity of a highly selected immunogenetic element.

Broadly neutralizing antibodies (bNAbs) are rarely elicited during HIV-1 infection. To identify obstacles to bNAb development, we longitudinally studied 122 rhesus macaques infected by one of 16 different simian-human immunodeficiency viruses (SHIVs). We identified V2 apex as the most common bNAb target and a subset of Envs that preferentially elicited these antibodies. In 10 macaques, we delineated Env-antibody coevolution from B cell priming to bNAb development. Antibody phylogenies revealed permissive developmental pathways guided by evolving Envs that contained few mutations in or near the V2 apex C-strand, which were a sensitive indicator of apex-targeted responses. The absence of such mutations reflected a failure in bNAb priming. These results indicate that efficiency of B cell priming, and not complexities in Env-guided affinity maturation, is the primary obstacle to V2 apex bNAb elicitation in SHIV-infected macaques and identify specific HIV-1 Envs to advance as novel vaccine platforms. One sentence summary:B cell priming is the primary bottleneck to HIV-1 V2 apex bNAb elicitation.

Local intramuscular administration of synthetic plasmid DNA (pDNA) encoding monoclonal antibodies (mAb) offers an alternative to recombinant protein-based mAb delivery. In this phase 1 dose-escalation study, we evaluated the safety, tolerability and pharmacokinetics of a pDNA cocktail encoding AZD5396 and AZD8076, modified versions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing mAb cocktail tixagevimab/cilgavimab in healthy adults. Participants received up to four intramuscular doses of pDNA encoding both DNA-based mAbs (DMAbs), administered using CELLECTRA electroporation. The primary endpoints were safety and pharmacokinetics. All 44 participants received at least one dose; DMAbs were detected in 100% of evaluable participants (n = 39), with serum concentrations reaching a peak of 1.61 µg ml-1. Sustained expression was observed in all participants during the 72 weeks of follow-up. The study product was well tolerated, with no product-related serious adverse events reported. Exploratory analyses demonstrated binding to multiple SARS-CoV-2 Spike protein variants and neutralizing activity in a standard pseudovirus assay. No antidrug antibodies were detected across approximately 1,000 serum samples using validated tiered assays. To our knowledge, these data represent the first-in-human proof-of-concept that synthetic pDNA DMAb technology permits the durable in vivo production of a functional mAb cocktail. This study further underscores the collective importance of synthetic design, formulation and delivery to achieve biologically relevant expression of gene-encoded biologics. DMAb delivery may represent a long-acting, scalable, cold-chain-independent platform against a wide range of diseases that can be targeted with mAbs and their derivatives. ClinicalTrials.gov registration: NCT05293249.

One approach for an HIV vaccine is through broadly neutralizing antibody (bnAb) elicitation. However, a major challenge with this approach is that bnAb precursors are still largely unknown. Here, we propose combining bioinformatics, deep learning structure prediction, and molecular dynamics simulations to identify and better understand potential precursors in the human antibody repertoire. We conducted various searches within an antibody database to identify potential precursors and then used deep learning structure prediction and simulations to isolate the most promising precursors.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Monoclonal antibodies are an important class of biologics with over 160 Food and Drug Administration/European Union-approved drugs. A significant bottleneck to global accessibility of recombinant monoclonal antibodies stems from complexities related to their production, storage, and distribution. Recently, gene-encoded approaches such as mRNA, DNA, or viral delivery have gained popularity, but ensuring biologically relevant levels of antibody expression in the host remains a critical issue. Using a synthetic DNA platform, we investigated the role of antibody structure and sequence toward in vivo expression. SARS-CoV-2 antibody 2196 was recently engineered as a DNA-encoded monoclonal antibody (DMAb-2196). Utilizing an immunoglobulin heavy and light chain "chain-swap" methodology, we interrogated features of DMAb-2196 that can modulate in vivo expression through rational design and structural modeling. Comparing these results to natural variation of antibody sequences resulted in development of an antibody frequency score that aids in the prediction of expression-improving mutations by leveraging antibody repertoire datasets. We demonstrate that a single amino acid mutation identified through this score increases in vivo expression up to 2-fold and that combinations of mutations can also enhance expression. This analysis has led to a generalized pipeline that can unlock the potential for in vivo delivery of therapeutic antibodies across many indications.

Carbohydrates and glycoproteins modulate key biological functions. However, experimental structure determination of sugar polymers is notoriously difficult. Computational approaches can aid in carbohydrate structure prediction, structure determination, and design. In this work, we developed a glycan-modeling algorithm, GlycanTreeModeler, that computationally builds glycans layer-by-layer, using adaptive kernel density estimates (KDE) of common glycan conformations derived from data in the Protein Data Bank (PDB) and from quantum mechanics (QM) calculations. GlycanTreeModeler was benchmarked on a test set of glycan structures of varying lengths, or “trees”. Structures predicted by GlycanTreeModeler agreed with native structures at high accuracy for both de novo modeling and experimental density-guided building. We employed these tools to design de novo glycan trees into a protein nanoparticle vaccine to shield regions of the scaffold from antibody recognition, and experimentally verified shielding. This work will inform glycoprotein model prediction, glycan masking, and further aid computational methods in experimental structure determination and refinement.

Abstract An alluring strategy for improving protective humoral immunity against infectious diseases would be to directly target the germinal center (GC). Robust GC responses are associated with high titer and high affinity antibody responses. Despite advances in formulation strategies and vaccine design, there remains a critical need for next generation approaches to elicit potent humoral immunity. Multimeric nanoparticles offer enhanced immunogenicity compared with monomeric antigen partially due to potent B cell activation and increased antigen density on the nanoparticle. Here, we investigated whether GC cytokine IL-21 could be displayed on nanoparticles to further enhance immune responses. We designed a chimeric nanoparticle using the GT8-60mer, a germline targeting HIV immunogen, as a model to scaffold IL-21. GT8 and IL-21 scaffolded nanoparticles, termed GT8 IL-21 nanoparticle immunoadjuvant complexes (GT8 IL-21 NIC), increased antigen-specific GC B cells, functional Tfh cell responses, and serum antibody titers relative to the antigen-only GT8-60mer. Additionally, GT8 IL-21 NICs reorganized antigen-specific GC B cell responses to the light zone (LZ) of the GC, the canonical site of antigen-driven selection. Single-cell RNA sequencing of antigen-specific GC B cells from GT8 IL-21 NIC immunized mice demonstrated upregulation of selection-associated gene signatures and a shift in the clonal repertoire. Thus, GC targeting NICs may provide value to drive improved humoral immunity.

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

COVID-19 remains a major public health concern. Monoclonal antibodies have received emergency use authorization (EUA) for pre-exposure prophylaxis against COVID-19 among high-risk groups for treatment of mild to moderate COVID-19. In addition to recombinant biologics, engineered synthetic DNA-encoded antibodies (DMAb) are an important strategy for direct in vivo delivery of protective mAb. A DMAb cocktail was synthetically engineered to encode the immunoglobulin heavy and light chains of two different two different Fc-engineered anti-SARS-CoV-2 antibodies. The DMAbs were designed to enhance in vivo expression and delivered intramuscularly to cynomolgus and rhesus macaques with a modified in vivo delivery regimen. Serum levels were detected in macaques, along with specific binding to SARS-CoV-2 spike receptor binding domain protein and neutralization of multiple SARS-CoV-2 variants of concern in pseudovirus and authentic live virus assays. Prophylactic administration was protective in rhesus macaques against signs of SARS-CoV-2 (USA-WA1/2020) associated disease in the lungs. Overall, the data support further study of DNA-encoded antibodies as an additional delivery mode for prevention of COVID-19 severe disease. These data have implications for human translation of gene-encoded mAbs for emerging infectious diseases and low dose mAb delivery against COVID-19.

Broadly neutralizing antibodies (bnAbs) have promise to protect against HIV infection, but induction of bnAbs by immunization is an unsolved vaccine design challenge. Germline-targeting priming immunogens aim to initiate the induction of bnAbs by specifically activating rare bnAb-precursor B cells that can subsequently be matured using suitable heterologous boosting and shepherding immunogens. Several pre-clinical studies, and the IAVI G001 human clinical trial, have demonstrated the ability of a germline-targeting priming immunogen, eOD-GT8 60mer, to induce precursors of the VRC01 class of bnAbs. However, much less is known about B cells induced against other epitopes of the immunogen. Here, we performed unbiased analysis of B cells induced by eOD-GT8 60mers in Intelliselect Transgenic mice (Kymice) that are transgenic for the human Ig loci and produce human-like BCRs. B cells isolated with intact eOD-GT8 60mer nanoparticles showed a large diversity of non-VRC01-class B cells, with 38% unique clonotypes and only 5% of BCRs belonging to public lineages shared among all animals. We found that many competitors recognize epitopes in close proximity to or overlapping with the VRC01 epitope. These results indicate that optimal boosting of VRC01-class bnAb-precursor B cells primed by eOD-GT8 60mer might require a first-boost immunogen that minimizes recognition of competitor B cells, and such competitors isolated from Kymice could serve as valuable reagents for boost development. ### Competing Interest Statement Materials and information concerning the immunogens are available by material transfer agreement from the Scripps Research Institute. IAVI and the Scripps Research Institute have filed a patent (U.S. PCT Application no. PCT/US2016/038162) relating to the eOD-GT8 immunogens in this manuscript, which included inventors J.G.J., D.W.K., S.M., and W.R.S. The Kymab mouse strains described are corporate assets protected by multiple patents; access to these mice is available through licensing.

Many prokaryotes use swimming motility to move toward favorable conditions and escape adverse surroundings. Regulatory mechanisms governing bacterial flagella-driven motility are well-established; however, little is yet known about the regulation underlying swimming motility propelled by the archaeal cell surface structure, the archaella. Previous research showed that the deletion of the adhesion pilins (PilA1-6), subunits of the type IV pili cell surface structure, renders the model archaeon Haloferax volcanii non-motile. In this study, we used ethyl methanesulfonate mutagenesis and a motility assay to identify motile suppressors of the triangle pilA[1-6] strain. Of the eight suppressors identified, six contain missense mutations in archaella biosynthesis genes, arlI and arlJ. In trans expression of arlI and arlJ mutant constructs in the respective multi-deletion strains triangle pilA[1-6]triangle arlI and triangle pilA[1-6]triangle arlJ confirmed their role in suppressing the triangle pilA[1-6] motility defect. Additionally, three suppressors harbor co-occurring disruptive missense and nonsense mutations in cirA, a gene encoding a proposed regulatory protein. A deletion of cirA resulted in hypermotility, while cirA expression in trans in wild-type cells led to decreased motility. Moreover, quantitative real-time PCR analysis revealed that in wild-type cells, higher expression levels of arlI, arlJ, and the archaellin gene arlA1 were observed in motile early-log phase rod-shaped cells compared to non-motile mid-log phase disk-shaped cells. Conversely, triangle cirA cells, which form rods during both early- and mid-log phases, exhibited similar expression levels of arl genes in both growth phases. Our findings contribute to a deeper understanding of the mechanisms governing archaeal motility, highlighting the involvement of ArlI, ArlJ, and CirA in pilin-mediated motility regulation.