Biology subjects

Crispin, M.

Publications and source records attributed to Crispin, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Xenoglycosylation of the HIV-1 Glycan Shield Promotes Cross-Clade Neutralization and Diverse Human Antibody Lineages

Despite advances in HIV-1 envelope glycoprotein (Env)-based vaccine design, eliciting broadly neutralizing antibodies (bNAbs) remains a central challenge. Here, we identify core (1,3)-fucose as a key modulator of neutralizing antibody (NAb) induction and breadth. Uncleaved prefusion-optimized (UFO) trimers and self-assembling protein nanoparticles (SApNPs) produced in High Five (Hi5) insect cells incorporate core (1,3)-fucose into the Env glycan shield. Crystal structures of Hi5-produced BG505 UFO trimers in complex with bNAbs confirm native-like Env architecture and epitope presentation. In wild-type mice, these trimers and SApNPs elicit robust autologous and heterologous NAb responses. We then engineer Chinese hamster ovary (CHO) cells to co-express three insect- and plant-derived core (1,3)-fucosyltransferases. The resulting immunogens carry fucosylated mammalian glycans and elicit cross-clade NAb responses in mice, supporting an important role for core (1,3)-fucose in broadened neutralization. Env-specific IgE responses suggest engagement of associated immune pathways during vaccination. In ATX-Gx humanized mice, core (1,3)-fucosylated, glycan-trimmed Env elicits detectable cross-clade neutralization by week 11. Single-cell analysis identifies distinct human antibody lineages with cross-clade activity and noncanonical Env recognition. Repertoire and functional analyses uncover putatively related lineage members across two animals within the same group. Together, these findings support xenoglycosylation as a promising, germline-agnostic strategy for HIV-1 vaccine design.

immunology↗

bioRxiv2026

Programming N-linked glycan composition through de novo protein design

N-linked glycans are ubiquitous post-translational modifications that mediate diverse biological processes, including cell signaling and adhesion, molecular recognition, and host-pathogen interactions. Despite their importance, N-linked glycans have largely remained beyond the reach of biomolecular design due to a lack of methods capable of jointly modeling glycans and protein structure. Here, we develop a general all-atom computational pipeline for creating de novo glycoproteins in which the amino acid sequence encodes both the location and composition of N-linked glycans. The method uses three-dimensional protein structure to tune glycan accessibility to processing enzymes in the eukaryotic secretory pathway. Systematic increases in steric restriction progressively reduced processing, shifting glycans from heterogeneous complex-type structures toward increasingly underprocessed oligomannose-type species resembling those found on many viral glycoproteins. Despite this programmed restriction, the resulting oligomannose glycans remained accessible for biological recognition, inhibiting uropathogenic Escherichia coli adhesion to human bladder cells and binding mannose-binding lectin. These results establish steric control of N-glycan processing as a genetically encodable design principle and extend computational protein design to functional glycoproteins with programmed composition.

biochemistry↗

bioRxiv2026

Consensus native-like hepatitis C virus E1E2 engages broadly neutralizing antibody precursors

A major goal for hepatitis C virus (HCV) vaccine development is to elicit broadly neutralizing antibodies (bNAbs) against the E1E2 glycoprotein complex located on the viral surface. Inducing HCV bNAbs requires engagement of their germline B cell precursors. HCV glycoproteins usually do not bind and activate inferred germline precursors of bNAbs (igl-bNAbs), possibly because most circulating strains contain non-conserved isolate-specific residues, even in bNAb epitopes. Here, we generated stabilized native-like soluble E1E2 (sE1E2) antigens based on a consensus sequence of HCV (HepCon) to limit the exposure of antigenically rare residues. The antigenicity and glycosylation profiles show that HepCon sE1E2 resembles a native-like E1E2 heterodimer. HepCon sE1E2 induced cross-reactive neutralizing antibody responses as a soluble protein immunogen and as membrane-anchored mRNA-delivered immunogen in animals. Importantly, HepCon sE1E2 engages multiple igl-bNAbs against two major epitopes: antigenic region 3 (AR3), which is targeted by igl-bNAbs derived from the widely expressed human VH1-69 B cell gene, and antigenic region 4 (AR4), which is only present on native-like E1E2. Nanoparticles with HepCon sE1E2 efficiently activated B cell lines expressing AR3 and AR4 igl-bNAb B cell receptors in vitro. Finally, using HepCon sE1E2 we elucidated the atomic contacts of an AR3 igl-bNAb by cryo-electron microscopy. Thus, HepCon sE1E2 is a promising candidate for germline-targeting vaccination strategies.

immunology↗

bioRxiv2026

Extensive global HIV-1 sequence diversity perturbs conserved features of the envelope glycan shield

The extensive sequence diversity and glycosylation of the HIV-1 envelope glycoprotein pose a challenge for vaccine design efforts aimed at eliciting glycan-binding broadly neutralizing antibodies (bnAbs). Understanding how HIV-1 glycosylation varies across diverse strains is therefore of considerable interest. Here, we employed mass spectrometry to map the site-specific glycan composition of pseudoviruses from an established 12-virus panel representing global sequence diversity. We refine the current model of the viral glycan shield by showing how the presence or absence of glycans can modulate the composition and perimeter of the mannose patch, where glycans exhibit limited maturation. Importantly, we show that within the Clade A 398F1 strain, isolated in Tanzania, the trimer apex exhibited elevated glycan maturation and resistance to apex-directed bnAbs. These findings demonstrate that the 12-virus panel reveals both conserved glycan-dependent epitopes and strains with exceptional glycosylation features that may impact the generality of vaccine design efforts targeting particular epitopes.

biochemistry↗

bioRxiv2026

Translating Innovation to Clinic: End-to-End Bioprocess Development and cGMP Manufacturing of N332-GT5 HIV Vaccine Candidate for First-in-Human Trials HVTN144

The successful translation of rationally designed HIV-1 immunogens into effective vaccines requires manufacturing platforms that maintain structural conformity while meeting clinical-grade quality standards. We developed and scaled a robust, cGMP-compliant process for N332-GT5 gp140, a germline-targeting envelope trimer designed to initiate broadly neutralizing antibody responses, which is now undergoing first-in-human evaluation in HVTN144. Starting with a stable CHO cell line developed using Leap-In(R) transposon technology, we established a production clone exhibiting high-titer expression (>200 mg/L) and genetic stability through 60 population doublings. The manufacturing process scaled efficiently from Ambr(R) 250 miniature bioreactors to 200-L single-use systems, delivering consistent product quality across multiple cGMP batches. A streamlined three-step purification strategy--affinity capture, multimodal polishing, and viral clearance- yielded >99% trimeric purity with preserved quaternary structure and native-like antigenicity. Orthogonal LC-MS analyses confirmed site-specific glycan occupancy matching design specifications, while robust viral clearance exceeded 18-log and 11-log reductions for model retroviruses. Clinical material manufactured through this platform has been successfully administered in HVTN144. This work establishes a scalable, reproducible manufacturing paradigm for structurally complex HIV-1 envelope immunogens, advancing the field toward rational vaccine design based on germline-targeting principles.

bioengineering↗

bioRxiv2026

Decoding epitope immunodominance in HIV Env using cryoEM and machine learning

Viral surface glycoproteins, such as the HIV envelope protein (Env), present numerous antibody (Ab) epitopes, yet immune responses consistently focus on only a subset, a phenomenon known as immunodominance. Although structural studies have provided insights into Env antigenicity, our understanding of the molecular features that govern efficient Ab engagement remains incomplete, thereby limiting the predictive and rational design of vaccines. Here, we characterized the structural determinants of epitope immunodominance in HIV Env by integrating high-resolution cryoEM-based polyclonal epitope mapping (cryoEMPEM) across different clades with quantitative analyses of epitope topology, accessibility, and physicochemical properties. More than 70 new structures were resolved to assemble a library of >100 Env-antibody complexes. These data informed the development of a surface-centric, machine-learning model to predict relative Antigen Surface Immunodominance (ASI model). Comparison of ASI-predicted epitope sites with the specificities of Env-induced antibodies showed that the model accurately identifies immunodominant regions and highlights the structural features driving immune bias. Notably, immunogens redesigned based on model predictions successfully redirected Ab responses toward a normally subdominant epitope, demonstrating the potential of strategies coupling targeted assembly of focused structural libraries with machine learning to uncover complex molecular patterns and enable design of more effective vaccine antigens.

immunology↗

bioRxiv2025

Antibody feedback establishes an affinity brake in the germinal center

Recent advances in mRNA vaccine technology have opened the door to novel types of antigen display, but little is yet known as to how B cells recognize and respond to these formats. By delivering an mRNA-LNP encoded membrane-bound immunogen displaying three conserved HIV-1 Envelope (Env) epitopes to knock-in mouse models with B cell receptors (BCRs) of defined affinities, we investigated how epitope-specific competition shapes germinal center (GC) responses. Co-activation of B cells targeting different epitopes did not alter GC kinetics observed in individual activations, but a striking inverse correlation was observed between BCR affinity and GC residence time in either scenario: high-affinity B cells exhibited shorter persistence in GCs, while those with lower affinity to the antigen were maintained. Furthermore, B cells were able to engage in GC reactions at equivalent rates in the presence or absence of clonal lineages binding the same epitope with similar affinities, while higher-affinity clones suppressed lower-affinity counterparts targeting the same epitope. Spatial transcriptomics revealed plasma-like cells within and adjacent to the GC which, together with the detection of early IgG in draining lymph nodes, suggests that local antibody production from these cells may contribute to feedback-driven kinetics. These findings indicate that a self-modulated local antibody feedback loop may act as a "brake" on epitope-specific recognition--dampening further affinity enhancement for high-affinity B cells and facilitating epitope spreading by redirecting the response toward alternative epitopes.

immunology↗

bioRxiv2025

Structure-guided design of native-like HIV Env Single Chain Trimers for enhanced stability, immunogenicity, and versatile vaccine delivery

Stabilizing the HIV envelope (Env) trimer in its native prefusion conformation is key to eliciting broadly neutralizing antibodies (bnAbs). We present a generalizable single-chain trimer-(SCT) design platform that enables the production of stable, native-like Env trimers across diverse HIV strains. Using a structure-guided approach, we developed CAP256.SU-SCT9, based on a CAP256.SU Env shown to induce V2-apex bnAbs in humans and rhesus macaques. CAP256.SU-SCT9 trimer exhibited native-like structure, bnAb-specific antigenicity, authentic glycosylation, and compatibility with nucleic acid and nanoparticle delivery. We extended this design strategy to a panel of HIV Envs representing global diversity, and immunogens known to initiate bnAb responses in humans. Structural analyses confirmed that SCTs consistently maintained prefusion-closed conformations, favorable glycan profiles, strong binding to trimer-specific bnAbs, and low reactivity with non-neutralizing antibodies. These findings establish SCT as a versatile platform for HIV trimer immunogen design, with potential to support next-generation vaccines aimed at eliciting protective bnAb responses.

immunology↗

bioRxiv2025

Germline-targeting HIV immunogen induces cross-neutralizing antibodies in outbred macaques

Germline-targeting-(GT) is a promising strategy to activate rare broadly neutralizing antibody (bnAb)-producing B cells against HIV, but induction of such responses in outbred animals has not been achieved. Using antibody-guided structure-based design, we engineered a germline-targeting trimer immunogen Q23-APEX-GT2 that primes diverse V2-apex bnAb precursors. Q23-APEX-GT2 efficiently activated V2-apex-specific B cells in humanized knock-in mice and consistently elicited immunofocused antibody responses in rhesus macaques, priming multiple long CDRH3-loop bnAb-B cell lineages. Monoclonal antibodies from immunized macaques exhibited broad heterologous HIV trimer binding and cross-neutralization. Atomic-level structural studies confirmed precise epitope targeting and revealed CDRH3-paratope configurations that mirrored those of human V2-apex bnAbs. This study provides proof-of-principle for successful priming and maturation of authentic V2-apex bnAb precursors in outbred macaques, underscoring the potential of V2-apex-targeted vaccines. HIGHLIGHTSO_LIEngineered Q23-APEX-GT2 trimer to stimulate diverse V2-apex bnAb B cell precursors C_LIO_LIQ23-APEX-GT2 primed rare V2-apex bnAb B cells in mice and outbred rhesus macaques C_LIO_LIQ23-APEX-GT2 elicited immunofocused antibody responses and diverse V2-apex B cell lineages with desirable long-CDRH3 paratope properties C_LIO_LIQ23-APEX-GT2 alone induced V2-apex antibodies with broad HIV trimer binding and modest neutralization breadth C_LIO_LIStructural analysis confirmed bnAb site targeting, mirroring human and rhesus V2-apex bnAbs C_LI

immunology↗

bioRxiv2025

Regulation of N-glycosylation efficiency by eukaryotic oligosaccharyltransferase

Oligosaccharyltransferase (OST) catalyses the key step of N-glycosylation, transferring immature N-glycans to select Asn residues in nascent proteins in the endoplasmic reticulum (ER). Asn are more likely to be glycosylated in the context of a "glycosylation sequon", N-x-S/T (x=P), but not every sequon is glycosylated. Tight positive and negative regulation of site-specific N-glycosylation across the glycoproteome is essential because some Asn require glycosylation for productive protein folding or function, while others must remain unglycosylated. Despite its importance, the underlying bases for this regulation are illdefined. Here we characterise the molecular determinants regulating OST catalytic efficiency. We identify preferred substrates of OST, and show that the local sequence determines glycosylation efficiency by fine-tuning binding to an extended groove in OST along an eight residue stretch centered at the Asn acceptor. Tight control of OST activity is achieved through a switch, set ON when a peptide binds with high complementarity to the groove, triggering release of the assisting base Glu350 for catalysis. Based on analysis of buried sequons, we identify sequence characteristics associated with inherently non-preferred acceptor substrates for OST, and show these have a high propensity for local secondary structure incompatible with the optimal conformation of a peptide bound to OST. Finally, we validate these mechanisms by examining the determinants of efficient glycosylation at a variably glycosylated Asn in a set of human Fabs. These mechanistic and functional insights show how local protein sequence controls glycosylation occupancy with immediate implications in secretory protein function and evolution, pathogen immunity and epistasis, and in engineering protein sequences for the desired degree of site-specific glycosylation.

biochemistry↗

bioRxiv2025

Differential contributions of human oligosaccharyltransferase complexes OST-A and OST-B to HIV-1 envelope glycoprotein glycosylation

N-linked glycosylation of glycoproteins during synthesis in the endoplasmic reticulum (ER) is mediated by oligosaccharyltransferase (OST) complexes OST-A and OST-B that have different catalytic subunits STT3A and STT3B, respectively. OST-A acts cotranslationally, while OST-B adds glycans posttranslationally. While there is redundancy between these two enzymes, it is unclear how they both contribute to glycosylation of the densely glycosylated HIV-1 envelope glycoprotein complex (Env). We found that knocking out STT3A had a profound negative impact on HIV-1 virus production and infectivity while STT3B ablation had no such effect suggesting that STT3A is more important than STT3B for Env glycosylation and preserved function. STT3A/3B knockout (KO) affected the neutralization sensitivity to broadly neutralizing antibodies (bNAbs) in a strain-specific manner with STT3A-KO increasing susceptibility to VRC01 bNAb for the tested HIV-1 strains. In contrast, for the BG505 strain A virus, it conferred increased resistance to glycan-dependent bNAbs 2G12 and PGT128. For other HIV-1 strains, STT3B-KO also led to resistance to glycan-dependent bNAb PGT151. Site-specific glycan analysis of recombinant Env proteins revealed that STT3A-KO reduced glycan occupancy of potential N-linked glycosylation sites (PNGS) more globally than STT3B-KO, with certain acceptor sites, including N234 and N386, showing STT3A dependence. In contrast, STT3B-KO appeared to have a more pronounced effect on gp41 glycosylation, suggesting that PNGS located near the C-terminus are more dependent on STT3B. Defining the roles of the OST-A and OST-B complexes in HIV-1 Env glycosylation may bring critical information for the development of methods to control PNGS glycan occupancy of recombinant glycoprotein immunogens.

microbiology↗

bioRxiv2025

From sequence to scaffold: computational design of protein nanoparticle vaccines from AlphaFold2-predicted building blocks

Self-assembling protein nanoparticles are being increasingly utilized in the design of next-generation vaccines due to their ability to induce antibody responses of superior magnitude, breadth, and durability. Computational protein design offers a route to novel nanoparticle scaffolds with structural and biochemical features tailored to specific vaccine applications. Although strategies for designing new self-assembling proteins have been established, the recent development of powerful machine learning-based tools for protein structure prediction and design provides an opportunity to overcome several of their limitations. Here, we leveraged these tools to develop a generalizable method for designing novel self-assembling proteins starting from AlphaFold2 predictions of oligomeric protein building blocks. We used the method to generate six new 60-subunit protein nanoparticles with icosahedral symmetry, and single-particle cryo-electron microscopy reconstructions of three of them revealed that they were designed with atomic-level accuracy. To transform one of these nanoparticles into a functional immunogen, we reoriented its termini through circular permutation, added a genetically encoded oligomannose-type glycan, and displayed a stabilized trimeric variant of the influenza hemagglutinin receptor binding domain through a rigid de novo linker. The resultant immunogen elicited potent receptor-blocking and neutralizing antibody responses in mice. Our results demonstrate the practical utility of machine learning-based protein modeling tools in the design of nanoparticle vaccines. More broadly, by eliminating the requirement for experimentally determined structures of protein building blocks, our method dramatically expands the number of starting points available for designing new self-assembling proteins. Significance StatementSelf-assembling protein nanoparticle vaccines have steadily gained traction in both academic and industry vaccine development over the last decade. Recent work has shown that computationally designing new self-assembling proteins allows the structural and functional features of nanoparticle vaccines to be precisely tailored, and that this can significantly affect the vaccine-elicited immune response. To date, designing such nanoparticle vaccines has required the use of known crystal structures as starting points. Here we show how new machine learning tools can be leveraged to design new self-assembling protein nanoparticle immunogens in the absence of experimentally determined structures of the building blocks that elicit strong immune responses in mice.

biochemistry↗

bioRxiv2025

Rapid acquisition of HIV-1 neutralization breadth in a rhesus V2 apex germline antibody mouse model after a single bolus immunization

Current vaccine strategies to elicit broadly neutralizing antibodies (bnAbs) against HIV-1 generally propose complex, multi-boost immunization regimens. In rhesus macaques, SHIV infection has been observed to rapidly drive the development of some classes of bnAbs that share structural similarities with those in humans. Here, we generated a knockin mouse model with B cells bearing the unmutated common ancestor (UCA) of the V2 apex-targeted bnAb lineage, V033-a. A single immunization of mice with a germline-targeting native-like trimer was sufficient to recapitulate the ontogeny of the mature rhesus bnAb in knockin mice--including rare, disfavored somatic mutations--leading to the induction of antibodies that exhibited potent neutralization against both autologous and heterologous tier 2 viruses. A boost with Env escape mutant trimers further improved breadth and potency, and cryo-EM structure revealed the structural basis for heterologous neutralization breadth. Non-human primate and mouse models can thus combine with structure to serve as a platform for identifying and confirming immunogens that streamline HIV-vaccination regimens.

immunology↗

bioRxiv2025

Native-like soluble E1E2 glycoprotein heterodimers on self-assembling protein nanoparticles for hepatitis C virus vaccine design

Hepatitis C virus (HCV) is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. E1E2-based HCV vaccine development has been hindered by the challenge of producing a soluble E1E2 (sE1E2) antigen that faithfully recapitulates the native glycoprotein heterodimer found on virions. Based on available cryo-electron microscopy (cryo-EM) structures, we rationally engineered sE1E2 for genotype 1a H77 by truncating the E1 and E2 stems (Cut1), removing a putative fusion peptide (pFP)-containing region in E1 (Cut2), and stabilizing the E1-E2 interface with diverse heterodimeric scaffolds. All H77 sE1E2.Cut1+2 scaffolds showed native-like E1-E2 association and robust binding to the broadly neutralizing antibody (bNAb) AR4A. A genotype 1a HCV-1 sE1E2.Cut1+2 variant scaffolded by a modified SpyTag/SpyCatcher (SPY{Delta}N) was selected for in vitro, structural, and immunogenic characterization. The structure of this sE1E2 scaffold in complex with bNAbs was analyzed by cryo-EM and negative-stain EM (nsEM), with an nsEM-based approach developed for antibody epitope mapping. HCV-1 sE1E2.Cut1+2.SPY{Delta}N was displayed on self-assembling protein nanoparticles (SApNPs) to enhance immunogenicity. HCV-1 sE1E2.Cut1+2.SPY{Delta}N heterodimer and SApNPs with wildtype and modified glycans were tested in mice, revealing the beneficial effects of multivalent display and oligomannose enrichment. Our study provides a rigorous foundation for next-generation HCV vaccine development. ONE-SENTENCE SUMMARYRational design, characterization, and in vivo assessment of HCV soluble E1E2 heterodimer and nanoparticles will inform vaccine development.

microbiology↗

bioRxiv2025

Harnessing mRNA for the expression of monoclonal IgG and IgA in non-human primates.

Monoclonal antibodies (mAbs) are an increasingly essential class of medicines across many disease areas (1). In the human body, there are five antibody isotypes, each with potential prophylactic or therapeutic benefits for different disease indications. However, 97% of all clinically approved mAbs are produced as the IgG isotype, partly due to differences in half-life, but largely due to challenges associated with recombinantly producing non-IgG isotypes like IgM or IgA, which have additional N-linked glycan sites and can present as multivalent oligomers. One potential solution to this challenge is to express mAbs in situ using mRNA encapsulated in lipid nanoparticles (LNP), bypassing the need for recombinant protein production (2). Here, we demonstrate the feasibility of expressing a mAb as both IgG and IgA in non-human primates (NHPs) using mRNA-LNPs. We express ePGDM1400v9, a broadly neutralizing mAb targeting human immunodeficiency virus (HIV), in both IgG1 and IgA2 formats by infusing NHPs with LNPs containing the appropriate mRNA. Though IgG1 expression levels were higher than those of IgA2, both formats were detectable in serum within one day of LNP infusion in all NHPs, and both were detectable in mucosal secretions of most animals. Importantly, serum mRNA-produced IgG1 and IgA2 retained HIV-neutralizing function. Furthermore, mass spectrometry analysis confirmed that mAbs of either isotype produced in situ exhibited glycosylation patterns highly similar to that of native antibody, which is likely to confer therapeutic advantages. Altogether, this work demonstrates the feasibility of using mRNA-LNPs to express native-like mAbs of non-IgG isotypes in primates and enables further development of non-IgG mAb constructs. SignificanceMonoclonal antibodies (mAbs) are a rapidly growing class of essential medicines across diverse disease areas and applications. However, the impact potential of mAbs is limited by challenges in production and purification, which favors the use of the IgG antibody isotype despite the disease-specific advantages that other isotypes might offer. One way to overcome this challenge is to express mAbs in situ using messenger ribonucleic acid delivered by lipid nanoparticles (mRNA-LNPs). Here, we show that an anti-human immunodeficiency virus (HIV) mAb, ePGDM1400v9, can be expressed as two different antibody isotypes, IgG and IgA, in nonhuman primates (NHPs) by mRNA-LNP delivery. We demonstrate that both mAb isotypes retain function and exhibit native-like glycosylation patterns that are not achievable with conventional recombinant mAbs.

immunology↗

bioRxiv2025

The Origin, Diagnosis, and Prognosis of Oligomannose-Type Diffuse Large B Cell Lymphoma

The acquisition of N-glycosylation sites occupied by oligomannose-type glycans in the immunoglobulin complementarity-determining region (CDR) is an early clonal tumor-specific identifier of follicular lymphoma (FL). CDR-located N-glycosylation sites are also acquired in germinal-center-B-cell-like diffuse large B-cell lymphomas (GCB-DLBCL), but their significance is less defined. We used RNA-seq immunoglobulin assembly to determine the frequency and CDR location of the acquired N-glycosylation sites (AGS) in two large independent DLBCL cohorts. Composition of the glycans occupying the AGS was determined using liquid chromatography-mass spectrometry and correlated with cell-of-origin, FL signature (defined by EZB phenotype or BCL2 translocation), transcript profile, and clinical outcome. CDR-located AGS were observed in 41-46% of GCB-DLBCL but were rare in other DLBCL. Only CDR-located AGS of DLBCL with an FL signature were occupied by oligomannose-type glycans. These DLBCL were termed Mann-type DLBCL. Conversely, the AGS of the other DLBCL were either non-glycosylated or occupied by complex-type glycans. Mann-type status was an independent marker of short progression-free survival and overall survival. In contrast, the other GCB-DLBCL cases, including those with an FL signature but without AGS, had the best outcomes. Mann-type DLBCL overexpressed gene-sets of cell growth, survival, and cycling, and underexpressed proinflammatory and apoptotic pathways, irrespective of concomitant MYC translocations. Acquisition of Mann-type glycans is a highly selective environmental pressure, identifying the most aggressive GCB-DLBCL with an origin related to FL. The detection of AGS in the CDR of GCB-DLBCLs with a BCL2 translocation defines Mann-type DLBCLs, refines prognosis and marks a precise tumor interaction to block early therapeutically. Key pointsO_LIAcquisition of Mann-type glycans is a tumor-specific environmental pressure identifying the most aggressive GCB-DLBCL with an origin related to FL. C_LIO_LIMann-type DLBCL can be diagnosed by the concomitant detection of AGS in the CDR and a BCL2 translocation. C_LI

cancer biology↗

bioRxiv2025

Rational design of next-generation filovirus vaccines with glycoprotein stabilization, nanoparticle display, and glycan modification

Filoviruses pose a significant threat to human health due to frequent outbreaks and high mortality. Although two vector-based vaccines are available for Ebola virus, a broadly protective filovirus vaccine remains elusive. Here, we evaluate a general strategy for stabilizing glycoprotein (GP) structures from Ebola, Sudan, and Bundibugyo orthoebolaviruses and Ravn orthomarburgvirus. A 3.2 [A] crystal structure provides atomic-level details of the redesigned Ebola virus GP, while cryo-electron microscopy reveals how a pan-orthoebolavirus neutralizing antibody targets a conserved site on the stabilized Sudan virus GP (3.13 [A] resolution), along with a low-resolution model of antibody-bound Ravn virus GP. A self-assembling protein nanoparticle (SApNP), I3-01v9, is redesigned at the N terminus to enable optimal surface display of filovirus GP trimers. Following detailed in vitro characterization, we examine the lymph node dynamics of Sudan virus GP and GP-presenting SApNPs in mice. Compared with the soluble trimer, SApNPs exhibit [~]112-fold longer retention in lymph node follicles, up to 28-fold greater presentation on follicular dendritic cell dendrites, and up to 3-fold stronger germinal center reactions. Functional antibody responses induced by filovirus GP trimers and SApNPs bearing wild-type and modified glycans are assessed in mice. This study provides a foundation for next-generation filovirus vaccine development. ONE-SENTENCE SUMMARYFilovirus glycoproteins and nanoparticles were rationally designed and characterized in vitro and in vivo to aid filovirus vaccine development.

microbiology↗

bioRxiv2024

Immunofocusing on the conserved fusion peptide of HIV envelope glycoprotein in rhesus macaques

SummaryDuring infection, the fusion peptide (FP) of HIV envelope glycoprotein (Env) serves a central role in viral fusion with the host cell. As such, the FP is highly conserved and therefore an attractive epitope for vaccine design. Here, we describe a vaccination study in non-human primates (NHPs) where glycan deletions were made on soluble HIV Env to increase FP epitope exposure. When delivered via implantable osmotic pumps, this immunogen primed immune responses against the FP, which were then boosted with heterologous trimers resulting in a focused immune response targeting the conserved FP epitope. Although autologous immunizations did not elicit high affinity FP-targeting antibodies, the conserved FP epitope on a heterologous trimer further matured the lower affinity, FP-targeting B cells. This study suggests using epitope conservation strategies on distinct Env trimer immunogens can focus humoral responses on desired neutralizing epitopes and suppress immune-distracting antibody responses against non-neutralizing epitopes.

immunology↗