Biology subjects

Newby, M. L.

Publications and source records attributed to Newby, M. L..

12 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

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↗

bioRxiv2025

Consistent Induction of Broadly Neutralizing HIV Antibodies by a Novel Two-Step Mechanism Informs Immunogen Design

A major obstacle confronting HIV-1 vaccine and cure research is the lack of an outbred animal model for rapid and consistent induction of broadly neutralizing antibodies (bNAbs). We designed an epitope-focused simian-human immunodeficiency virus (SHIV.5MUT) that elicited broad and potent V3-glycan-targeted antibodies within a year of infection in 14 of 22 macaques compared with 0 of 14 control animals. SHIV.5MUT elicited bNAbs by a novel two-step mechanism, inducing an initial wave of V1-directed antibodies that selected for Envs with shortened, hypoglycosylated V1 loops, which in turn primed V3-glycan bNAb precursors. Rhesus bNAbs were immunogenetically and structurally diverse, closely resembling human V3-glycan bNAbs. Env-bNAb coevolution revealed a diverse repertoire of bNAb precursors and the Env variants that matured them, yielding a molecular blueprint for vaccine design.

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

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

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

Design and immunogenicity of an HIV-1 clade C pediatric envelope glycoprotein stabilized by multiple platforms

Various design platforms are available to stabilize soluble HIV-1 envelope (Env) trimers, which can be used as antigenic baits and vaccine antigens. However, stabilizing HIV-1 clade C trimers can be challenging. Here, we stabilized an HIV-1 clade C trimer based on an Env isolated from a pediatric elite-neutralizer (AIIMS_329) using multiple platforms, including SOSIP.v8.2, ferritin nanoparticles (NP) and an I53-50 two-component NP, followed by characterization of their biophysical, antigenic, and immunogenic properties. The stabilized 329 Envs showed binding affinity to trimer-specific HIV-1 broadly neutralizing antibodies (bnAbs), with negligible binding to non-neutralizing antibodies (non-nAbs). Negative-stain electron microscopy (nsEM) confirmed the native-like conformation of the Envs. Multimerization of 329 SOSIP.v8.2 on ferritin and two-component I53-50 NPs improved the overall affinity to HIV-1 bnAbs and immunogenicity in rabbits. These stabilized HIV-1 clade C 329 Envs demonstrate the potential to be used as antigenic baits and as components of multivalent vaccine candidates in future.

immunology↗

bioRxiv2024

Different vaccine platforms result in distinct antibody responses to the same antigen in haemodialysis patients

Generalised immune dysfunction in chronic kidney disease, especially in patients requiring haemodialysis (HD), significantly enhances the risk of severe infections. Moreover, vaccine-induced immunity is typically reduced in HD populations, but the full mechanisms behind this remain unclear. The SARS-CoV-2 pandemic provided an opportunity to examine the magnitude and functionality of antibody responses in HD patients to a previously unencountered antigen, Spike (S)-glycoprotein, after vaccination with different vaccine platforms (viral vector (VV); mRNA (mRV)). Here, we compared total and functional anti-S antibody responses (cross-variant neutralisation and complement binding) in 187 HD patients and 43 healthy controls 21-28 days after serial immunisation. After 2 doses of the same vaccine, HD patients had anti-S antibody levels and complement binding capacity comparable to controls. However, 2 doses of mRV induced greater polyfunctional antibody responses than VV, yet previous SARS-CoV-2 infection or an mRV boost after 2 doses of VV significantly enhanced antibody functionality in HD patients. Therefore, HD patients can generate near-normal, functional antigen-specific antibody responses following serial vaccination to a novel antigen, suggesting largely intact B cell memory. Encouragingly, exploiting immunological memory by using mRNA vaccines and boosting may improve the success of vaccination strategies in this vulnerable patient population.

immunology↗

bioRxiv2022

Structural conservation of Lassa virus glycoproteins and recognition by neutralizing antibodies

SummaryLassa fever is an acute hemorrhagic fever caused by the zoonotic Lassa virus (LASV). The LASV glycoprotein complex (GPC) mediates viral entry and is the sole target for neutralizing antibodies. Immunogen design is complicated by the metastable nature of recombinant GPCs and the antigenic differences amongst LASV lineages. Despite the sequence diversity of GPC, structures of most lineages are lacking. We present the development and characterization of prefusion-stabilized, trimeric GPCs of LASV lineages II, V, and VI, revealing structural conservation despite sequence diversity. High-resolution structures and biophysical characterization of GPC in complex with GP1-A antibodies reveal their neutralization mechanisms. Finally, we present the isolation and characterization of a novel trimer-preferring neutralizing antibody belonging to the GPC-B competition group with an epitope that spans adjacent protomers and includes the fusion peptide. Our work provides molecular detail information on LASV antigenic diversity and will guide efforts to design pan-LASV vaccines. HighlightsO_LIStructural characterization of soluble glycoproteins from four Lassa virus lineages. C_LIO_LIMAb 12.1F, belonging to the GP1-A cluster, inhibits matriglycan and LAMP-1 binding. C_LIO_LIGP1-A mAbs show glycan-dependence with 19.7E demonstrating lineage-dependent binding. C_LIO_LIA novel trimer-preferring NAb S370.7 targets the GPC-B epitope. C_LI

immunology↗

bioRxiv2022

Natural variations within the glycan shield of SARS-CoV-2 impact viral spike dynamics

The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity, whether arisen naturally or through vaccination. Understanding the structure of the viral spike assists in determining the impact of mutations on the antigenic surface. One class of mutation impacts glycosylation attachment sites, which have the capacity to influence the antigenic structure beyond the immediate site of attachment. Here, we compare the glycosylation of a recombinant viral spike mimetic of the P.1 (Gamma) strain, which exhibits two additional N-linked glycan sites compared to the equivalent mimetic of the Wuhan strain. We determine the site-specific glycosylation of these variants and investigate the impact of these glycans by molecular dynamics. The N188 site is shown to exhibit very limited glycan maturation, consistent with limited enzyme accessibility. Structural modeling and molecular dynamics reveal that N188 is located within a cavity by the receptor binding domain, which influences the dynamics of these attachment domains. These observations suggest a mechanism whereby mutations affecting viral glycosylation sites have a structural impact across the antigenic surface.

molecular biology↗