Biology subjects

Berndsen, Z. T.

Publications and source records attributed to Berndsen, Z. T..

11 recordsLinked to original sources

bioRxiv2025

Parenteral Vaccination with recombinant EtpA glycoprotein impairs enterotoxigenic E. coli colonization

Enterotoxigenic E. coli (ETEC) cause hundreds of millions of cases of acute diarrheal illness in low-middle income regions, disproportionately in young children. To date there is no licensed, broadly protective vaccine to protect against these common but antigenically heterogeneous pathogens. One of the more highly conserved antigens of ETEC, EtpA, is an extracellular glycoprotein adhesin that preferentially binds to blood group A glycans on intestinal epithelia. EtpA contributes to increased severity of illness in blood group A individuals, elicits robust serologic and fecal antibody responses following infection, and has been associated with protection against subsequent infection. However, its utility as a protective antigen needs further examination. In the present studies we examined whether parenteral vaccination with recombinant EtpA (rEtpA) could afford protection against intestinal colonization in a murine model of ETEC infection. Here, we demonstrate that intramuscular vaccination with rEtpA when adjuvanted with double mutant LT (dmLT) primes IgG predominant mucosal antibody responses to ETEC challenge. Notably, however, both antibody levels and avidity, as well as protection were dependent on vaccination schedule. Likewise, by electron microscopy polyclonal epitope mapping (EMPEM) we observed a greater diversity of epitopes targeted by antibodies after a more protracted vaccination schedule. Next, we explored the utility of IM immunization with alum-adjuvanted rEtpA. This elicited strong serologic and fecal IgG responses. Although accompanied by negligible IgA mucosal responses, EtpA alum-adjuvanted IM vaccination nevertheless protected against ETEC intestinal colonization. Collectively, these data suggest that EtpA could expand the portfolio of antigens targeted in ETEC subunit vaccine development.

microbiology↗

bioRxiv2024

Structural and immunological characterization of the H3 influenza hemagglutinin during antigenic drift

The quest for a universal influenza vaccine holds great promise for mitigating the global burden of influenza-related morbidity and mortality. However, challenges persist in identifying conserved epitopes capable of inducing protection. In this study, we explore the influence of glycan evolution on H3 hemagglutinin from 1968 to present day and its impacts on antigenicity and immunogenicity. We observe that the appearance of potential N-linked glycosylation sites in Sing/16 hemagglutinin head domain reduces the binding of broadly neutralizing antibodies and shifts the polyclonal immune response upon vaccination to target the stem. Furthermore, structural characterization of HK/68 and Sing/16 by cryo-electron microscopy shows that while HK/68 is resistant to enzymatic deglycosylation, removal of glycans destabilizes the hyperglycosylated head and membrane-proximal region in Sing/16. These insights expand our understanding of glycans beyond their role in protein folding and highlight the interplay among glycan integration and immune recognition to design a universal influenza vaccine.

immunology↗

bioRxiv2024

Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as human volunteers challenged with ETEC, diarrheal severity is significantly increased severity in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of human volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected human volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design. Author summaryEnterotoxigenic E. coli (ETEC), a leading cause of diarrhea disproportionately affecting young children in low-income regions, are a priority for vaccine development. Individuals possessing A blood-type are more susceptible to severe cholera-like disease. EtpA, a secreted, immunogenic, blood group A binding protein, is a current vaccine target antigen. Here, we determined the atomic structure of EtpA in complex with protective as well as non-protective monoclonal antibodies targeting two different domains of the protein, allowing us to pinpoint key regions involved in blood-group A antigen recognition and uncover the mechanism of antibody-based protection. In addition, we show through mass-spectrometry that EtpA is extensively and heterogeneously glycosylated at surface-exposed asparagine residues by a promiscuous and low-fidelity glycosyltransferase, EtpC, and that this unique form of bacterial glycosylation is critical for to development of protective immune responses. Lastly, polyclonal antibodies from vaccinated mice as well as monoclonal antibodies obtained from ETEC-infected human volunteers revealed that the highly antigenic surface of EtpA exhibits both protective and non-protective epitopes. These results greatly expand our understanding of ETEC pathogenesis, and the immune responses elicited by these common infections, providing valuable information to aid in the rational design and testing of subunit vaccines.

microbiology↗

bioRxiv2024

The Structure of ApoB100 from Human Low-density Lipoprotein

Low-density lipoprotein (LDL) plays a central role in lipid and cholesterol metabolism and is a key molecular agent involved in the development and progression of atherosclerosis, a leading cause of mortality worldwide. Apolipoprotein B100 (apoB100), one of the largest proteins in the genome, is the primary structural and functional component of LDL, yet its size and complex lipid associations have posed major challenges for structural studies. Here we overcome those challenges and present the first structure of apoB100 from human LDL using an integrative approach of cryo-electron microscopy, AlphaFold2, and molecular dynamics-based refinement. The structure consists of a large globular N-terminal domain that leads into a [~]58 nm long x 4 nm wide continuous amphipathic {beta}-sheet that wraps completely around the circumference of the particle, holding it together like a belt. Distributed symmetrically across the two sides of the {beta}-belt are 9 strategically located inserts that vary in size from [~]30-700 residues and appear to have diverse functions. The largest two form long flexible strings of paired amphipathic helices that extend across the lipid surface to provide additional structural support through specific long-range interactions. These results suggest a mechanism for how the various domains of apoB100 act in concert to maintain LDL shape and cohesion across a wide range of particle sizes. More generally, they advance our fundamental understanding of LDL form and function and will help accelerate the design of potential new therapeutics.

biophysics↗

bioRxiv2021

Molecular insights into antibody-mediated protection against the prototypic simian immunodeficiency virus

SIVmac239 infection of macaques is a favored model of human HIV infection. However, the SIVmac239 envelope (Env) trimer structure, glycan occupancy, and the targets and ability of neutralizing antibodies (nAbs) to protect against SIVmac239 remain unknown. Here, we report the isolation of SIVmac239 nAbs that recognize a glycan hole and the V1/V4 loop. A high-resolution structure of a SIVmac239 Env trimer-nAb complex shows many similarities to HIV and SIVcpz Envs, but with distinct V4 features and an extended V1 loop. Moreover, SIVmac239 Env has a higher glycan shield density than HIV Env that may contribute to poor or delayed nAb responses in SIVmac239-infected macaques. Passive transfer of a nAb protects macaques from repeated low dose intraveneous SIVmac239 challenge at serum titers comparable to those described for protection of humans against HIV infection. Our results provide structural insights for vaccine design and shed light on antibody-mediated protection in the SIV model.

immunology↗

bioRxiv2021

Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM

In BriefHerein, we evaluated the immunogenicity of several BG505 SOSIP-based HIV Env immunogens in the rhesus macaque animal model using a combination of serology and biophysical approaches. We applied electron cryo-microscopy for high-resolution mapping of elicited polyclonal antibody responses, which provided detailed insights into the binding modes of the most common classes of antibodies elicited by BG505 SOSIP immunogens as well as the critical differences in immunogenicity that can occur as a consequence of engineered stabilizing mutations and partial glycan occupancy at different sites. SummaryEngineered 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. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

immunology↗

bioRxiv2020

Vulnerabilities in coronavirus glycan shields despite extensive glycosylation

Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development has focussed on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein, which mediates receptor recognition and membrane fusion. Coronavirus S proteins are extensively glycosylated viral fusion proteins, encoding around 69-87 N-linked glycosylation sites per trimeric spike. Using a multifaceted structural approach, we reveal a specific area of high glycan density on MERS S that results in the formation of under-processed oligomannose-type glycan clusters, which was absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan density of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Consistent with the ability of the antibody-mediated immune response to effectively target and neutralize coronaviruses, we demonstrate that the glycans of coronavirus spikes are not able to form an efficacious high-density global shield to thwart the humoral immune response. Overall, our data reveal how differential organisation of viral glycosylation across class I viral fusion proteins influence not only individual glycan compositions but also the immunological pressure across the viral protein surface.

microbiology↗

bioRxiv2020

Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens

Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Nanoparticles of different sizes and geometries can be designed and combined with appropriate antigens to fit the requirements of different immunization strategies. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to the underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. Comparing the humoral responses elicited by ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle to the corresponding soluble protein revealed that multivalent presentation increased the proportion of the overall antibody response directed against autologous neutralizing Ab epitopes present on the ConM-SOSIP trimers. Author SummaryProtein constructs based on soluble ectodomains of HIV glycoprotein (Env) trimers are the basis of many current HIV vaccine platforms. Multivalent antigen display is one strategy applied to improve the immunogenicity of different subunit vaccine candidates. Here, we describe and comprehensively evaluate a library of de novo designed, protein nanoparticles of different geometries for their ability to present trimeric Env antigens. We found three nanoparticle candidates that can stably incorporate model Env trimer on their surface while maintaining its structure and antigenicity. Immunogenicity of the designed nanoparticles is assessed in vitro and in vivo. In addition to introducing a novel set of reagents for multivalent display of Env trimers, this work provides both guiding principles and a detailed experimental roadmap for the generation, characterization, and optimization of Env-presenting, self-assembling nanoparticle immunogens.

immunology↗

bioRxiv2019

A Network-based approach for Quantifying the Resilience and Vulnerability of HIV-1 Native Glycan Shield

Dense surface glycosylation on the HIV-1 envelope (Env) protein acts as a shield from the adaptive immune system. However, the molecular complexity and flexibility of glycans make experimental studies a challenge. Here we have integrated high-throughput atomistic modeling of fully glycosylated HIV-1 Env with graph theory to capture immunologically important features of the shield topology. This is the first complete all-atom model of HIV-1 Env SOSIP glycan shield that includes both oligomannose and complex glycans, providing results which are physiologically more relevant than the previous models with uniform glycosylation. This integrated approach including quantitative comparison with cryo-electron microscopy data provides hitherto unexplored details of the native shield architecture and its difference from the high-mannose glycoform. We have also derived a measure to quantify the shielding effect over the antigenic protein surface that defines regions of relative vulnerability and resilience of the shield and can be harnessed for rational immunogen design.

biophysics↗

bioRxiv2019

HIV-1 Envelope and MPER antibody structures in lipid assemblies

Structural and functional studies of HIV Env as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Thus, most structural studies have utilized soluble forms where the regions C-terminal to the ectodomain are deleted. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstituted a full-length Env clone into a nanodisc with MSP1D1 scaffold, complexed it with an MPER targeting antibody 10E8, and structurally defined the full quaternary epitope of 10E8 consisting of lipid, MPER and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapted the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with peptidisc scaffold.

immunology↗

bioRxiv2019

Visualization of the HIV-1 Env Glycan Shield Across Scales

The dense array of N-linked glycans on the HIV-1 Envelope Glycoprotein (Env), known as the "glycan shield", is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure-function analysis. Here we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM), computational modeling, and site-specific mass-spectrometry (MS) to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of inter-glycan interactions that drive the formation of higher-order structure within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. Importantly, this identified unforeseen changes in the glycan shield of Env obtained from expression in the same CHO cell line used for GMP production. Finally, by capturing the enzymatic deglycosylation of Env in a time-resolved manner we found that highly connected glycan clusters are resistant to digestion and help stabilize the pre-fusion trimer, suggesting the glycan shield may function beyond immune evasion. Significance StatementThe HIV-1 Env "glycan shield" masks the surface of the protein from immune recognition, yet intrinsic heterogeneity defies a typical structure-function description. Using a complementary approach of cryo-EM, computational modeling, and mass-spectrometry we show how heterogeneity and dynamics affect glycan shield structure across scales. Our combined approach facilitated the development of new cryo-EM data analysis methods and allowed for validation of models against experiment. Comparison of Env across a range of glycosylation states revealed how subtle differences in composition impact glycan shield structure and affect the accessibility of epitopes on the surface. Finally, time-resolved cryo-EM experiments uncovered how highly connected glycan clusters help stabilize the pre-fusion trimer, suggesting the glycan shield may function beyond immune evasion.

biophysics↗