Biology subjects

Byrne, P. O.

Publications and source records attributed to Byrne, P. O..

9 recordsLinked to original sources

bioRxiv2026

Structure-based design of soluble prefusion-stabilized herpes simplex virus type 2 glycoprotein B antigens

Herpes simplex virus type 2 (HSV-2) causes genital herpes through latent infection and periodic reactivation. Although antivirals alleviate symptoms, no prophylactic or therapeutic vaccines have been licensed. Glycoprotein B (gB) is a class III fusion protein that mediates entry by irreversibly transitioning from a metastable prefusion conformation to a stable postfusion conformation. Leveraging prior structure-based designs for human cytomegalovirus (HCMV) gB, we engineered amino acid substitutions in HSV-2 gB to stabilize its prefusion conformation. Cryo-EM of the engineered construct revealed a prefusion conformation and non-native dimers of gB trimers. Introduction of N-linked glycosylation sites resulted in the gB-G3 variant, which exhibited improved expression and reduced dimerization. Cryo-EM of gB-G3 bound to neutralizing antibodies yielded a 2.8 [A] resolution structure of the stabilized prefusion conformation in a closed state, which differs from the open states observed in recently published HSV gB structures. Both prefusion and postfusion gB variants were evaluated for immunogenicity in mice, delivered either as a protein subunit or mRNA vaccine. Each gB conformation elicited robust humoral and cellular responses. However, prefusion stabilization of gB did not improve neutralizing antibody titers relative to the postfusion construct, consistent with prior observations for HCMV gB. Collectively, these findings reveal insights into prefusion gB conformational dynamics, provide stabilized reagents for studying gB-directed immune responses and inform HSV-2 vaccine design.

microbiology↗

bioRxiv2025

Design, Structure, and Immunogenicity of a Soluble Prefusion-stabilized EBV gB Antigen

Epstein-Barr virus (EBV), the causative agent of mononucleosis, is linked to over 140,000 annual cancer-related deaths globally and increases the risk of multiple sclerosis by up to 32-fold. As a herpesvirus, EBV establishes lifelong infection, and over 90% of U.S. adults are EBV-seropositive. Despite its significant disease burden, no approved EBV vaccines or therapeutics exist. Among EBV envelope glycoproteins, the fusion protein (gB) is strictly required for epithelial and B cell infection. Using a combination of AlphaFold-guided modeling, rational design, and ThermoMPNN-informed optimization, we engineered a stabilized prefusion gB variant, D2C3. This construct incorporates two inter-protomeric disulfide bonds and three cavity-filling substitutions, resulting in a melting temperature of 54 {degrees}C. Cryo-EM analysis of this construct allowed us to determine the prefusion structure of EBV gB, providing insights into the structural transitions required to adopt the postfusion conformation. Murine immunizations and depletion studies with human sera suggested a trend toward improved functional immunogenicity of D2C3 compared to postfusion gB. Collectively, these studies define engineering principles to stabilize class III fusion proteins, provide reagents to interrogate the human antibody response to EBV gB, and lay a foundation for further studies to develop EBV gB-based vaccine candidates.

biochemistry↗

bioRxiv2024

A human cytomegalovirus prefusion-like glycoprotein B subunit vaccine elicits similar humoral immunity to that of postfusion gB in mice

Human cytomegalovirus (HCMV) is the leading infectious cause of birth defects. Despite the global disease burden, there is no FDA-approved HCMV vaccine. The most efficacious HCMV vaccine candidates to date have used glycoprotein B (gB), a class III viral fusion protein, in its postfusion form. While some viral fusion proteins have been shown to elicit stronger neutralizing responses in their prefusion conformation, HCMV prefusion-like and postfusion gB were recently shown to elicit antibodies with similar fibroblast neutralization titers in mice. We aimed to define and compare the specificity and functionality of plasma IgG elicited by distinct prefusion-like and postfusion gB constructs. Prefusion-like and postfusion gB elicited comparable IgG responses that predominantly mapped to AD-5 antigenic domain known to elicit neutralizing antibodies. Interestingly, postfusion gB elicited significantly higher plasma IgG binding to cell-associated gB and antibody dependent cellular phagocytosis than that of prefusion-like gB. The vaccines elicited comparable neutralization titers of heterologous HCMV strain AD169r in fibroblasts, yet neither elicited neutralizing titers against the vaccine-matched strain Towne in fibroblasts. Our data indicate that gB in this prefusion-like conformation elicits similar specificity and functional humoral immunity to that of postfusion gB, unlike certain class I viral fusion proteins which have been used as vaccine antigens. These findings deepen our understanding of the immune response elicited by class III fusion proteins and may inform further design and testing of conformationally-dependent herpesvirus glycoprotein vaccine candidates.

immunology↗

bioRxiv2024

Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been FDA-approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 [A] resolution cryo-electron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses. TeaserA structure-based design campaign leads to stabilization of the class III viral fusion protein from HCMV in a prefusion-like conformation.

molecular biology↗

bioRxiv2024

Hybrid immunity to SARS-CoV-2 arises from serological recall of IgG antibodies distinctly imprinted by infection or vaccination

We used plasma IgG proteomics to study the molecular composition and temporal durability of polyclonal IgG antibodies triggered by ancestral SARS-CoV-2 infection, vaccination, or their combination ("hybrid immunity"). Infection, whether primary or post-vaccination, mainly triggered an anti-spike antibody response to the S2 domain, while vaccination predominantly induced anti-RBD antibodies. Immunological imprinting persisted after a secondary (hybrid) exposure, with >60% of the ensuing serological response originating from the initial antibodies generated during the first exposure. We highlight one instance where hybrid immunity arising from breakthrough infection resulted in a marked increase in the breadth and affinity of a highly abundant vaccination-elicited plasma IgG antibody, SC27. With an intrinsic binding affinity surpassing a theoretical maximum (KD < 5 pM), SC27 demonstrated potent neutralization of various SARS-CoV-2 variants and SARS-like zoonotic viruses (IC50 [~]0.1-1.75 nM) and provided robust protection in vivo. Cryo-EM structural analysis unveiled that SC27 binds to the RBD class 1/4 epitope, with both VH and VL significantly contributing to the binding interface. These findings suggest that exceptionally broad and potent antibodies can be prevalent in plasma and can largely dictate the nature of serological neutralization. HIGHLIGHTS{blacksquare} Infection and vaccination elicit unique IgG antibody profiles at the molecular level {blacksquare}Immunological imprinting varies between infection (S2/NTD) and vaccination (RBD) {blacksquare}Hybrid immunity maintains the imprint of first infection or first vaccination {blacksquare}Hybrid immune IgG plasma mAbs have superior neutralization potency and breadth

immunology↗

bioRxiv2022

Activity of EGFR transmembrane region variants indicates specific transmembrane dimers are not required for EGFR activity

The Epidermal Growth Factor Receptor (EGFR) is a Receptor Tyrosine Kinase that mediates cell proliferation and differentiation events during development and maintenance of complex organisms. Formation of specific, ligand-dependent EGFR dimers is a key step in stimulating EGFR signaling, and crystal structures of active, dimeric forms of isolated EGFR extracellular regions and kinase domains have revealed much about how dimer interactions regulate EGFR activity. The nature and role of the transmembrane region in regulating EGFR activity remains less clear, however. Proposed roles for the transmembrane region range from nonspecific but energetically favorable interactions to specific transmembrane dimer conformations being associated with active, inactive, or activity-modulated states of EGFR. To investigate the role of specific transmembrane dimers in modulating EGFR activity we generated thirteen EGFR variants with altered transmembrane sequences designed to favor or disfavor specific types of transmembrane region interactions. We show using FRET microscopy that EGFR transmembrane regions have an intrinsic propensity to associate in mammalian cell membranes that is counteracted by the extracellular region. We show using cell-based assays that each of the EGFR transmembrane variants except the Neu variant, which results in constitutive receptor phosphorylation, is able to autophosphorylate and stimulate phosphorylation of downstream effectors Erk and Akt. Our results indicate that many transmembrane sequences, including polyleucine, are compatible with EGFR activity and provide no evidence for specific transmembrane dimers regulating EGFR function.

molecular biology↗

bioRxiv2022

DISTINCTIVE FEATURES OF THE RESPIRATORY SYNCYTIAL VIRUS PRIMING LOOP COMPARED TO OTHER NON-SEGMENTED NEGATIVE STRAND RNA VIRUSES

De novo initiation by viral RNA-dependent RNA polymerases often requires a polymerase priming residue, located within a priming loop, to stabilize the initiating NTPs. Polymerase structures from three different non-segmented negative strand RNA virus (nsNSV) families revealed putative priming loops in different conformations, and an aromatic priming residue has been identified in the rhabdovirus polymerase. In a previous study of the respiratory syncytial virus (RSV) polymerase, we found that Tyr1276, the L protein aromatic amino acid residue that most closely aligns with the rhabdovirus priming residue, is not required for RNA synthesis but two nearby residues, Pro1261 and Trp1262, were required. In this study, we examined the roles of Pro1261 and Trp1262 in RNA synthesis initiation. Biochemical studies showed that substitution of Pro1261 inhibited RNA synthesis initiation without inhibiting back-priming, indicating a defect in initiation. Biochemical and minigenome experiments showed that the initiation defect incurred by a P1261A substitution could be rescued by factors that would be expected to increase the stability of the initiation complex, specifically increased NTP concentration, manganese, and a more efficient promoter sequence. These findings indicate that Pro1261 of the RSV L protein plays a role in initiation, most likely in stabilizing the initiation complex. However, we found that substitution of the corresponding proline residue in a filovirus polymerase had no effect on RNA synthesis initiation or elongation. These results indicate that despite similarities between the nsNSV polymerases, there are differences in the features required for RNA synthesis initiation. Author SummaryRSV has a significant impact on human health. It is the major cause of respiratory disease in infants and exerts a significant toll on the elderly and immunocompromised. RSV is a member of the Mononegavirales, the non-segmented, negative strand RNA viruses (nsNSVs). Like other viruses in this order, RSV encodes an RNA dependent RNA polymerase, which is responsible for transcribing and replicating the viral genome. Due to its essential role during the viral replication cycle, the polymerase is a promising candidate target for antiviral inhibitors and so a greater understanding of the mechanistic basis of its activities could aid antiviral drug development. In this study, we identified an amino acid residue within the RSV polymerase that appears to stabilize the RNA synthesis initiation complex and showed that it plays a role in both transcription and RNA replication. However, the corresponding residue in a different nsNSV polymerase does not appear to play a similar role. This work reveals a key feature of the RSV polymerase but identifies differences with the polymerases of other related viruses.

microbiology↗

bioRxiv2020

Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to [~]10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 [A]-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.

microbiology↗

bioRxiv2020

Ligand-independent EGFR oligomers do not rely on the active state asymmetric kinase dimer

The human Epidermal Growth Factor Receptor (EGFR/ERBB1) is a Receptor Tyrosine Kinase (RTK) that forms active oligomers in response to ligand. Much evidence indicates that EGFR/ERBB1 forms oligomers in the absence of ligand, but the structure and physiological role of these ligand-independent dimers remain unclear. We use fluorescence microscopy to measure the oligomer stability and FRET efficiency for homo- and hetero-oligomers of fluorescent-protein labeled forms of EGFR and its paralog, Human Epidermal Growth Factor Receptor 2 (HER2/ERBB2) in vesicles derived from native cell membranes. Both receptors form ligand-independent oligomers at physiological plasma membrane concentrations. Mutations introduced in the EGFR kinase region at a key interface within the active state dimer alter the FRET efficiency within ligand-independent EGFR oligomers but do not affect their stability. These results indicate that ligand-independent EGFR oligomers do not require this interface and that the inactive state ensemble is distinct from the EGFR active state ensemble.

biophysics↗