Biology subjects

Schiffner, T.

Publications and source records attributed to Schiffner, T..

11 recordsLinked to original sources

bioRxiv2025

CryoEM Structures of Antibodies Elicited by Germline-Targeting HIV MPER Epitope-Scaffolds

Applying cryoEM to small protein complexes is usually challenging due to their lack of features for particle alignment. Here, we characterized antibody responses to 21 kDa HIV membrane-proximal external region germline-targeting (MPER-GT) immunogens through cryoEM by complexing them with 10E8 or Fabs derived from MPER-GT immunized animals. Distinct antibody-antigen interactions were analyzed using atomic models generated from cryoEM maps. Mutagenesis screening revealed off-target mAbs that do not compete with 10E8 bind non-MPER epitopes, and the two most dominant epitopes were verified by cryoEM. The structures of 10E8-class on-target Fabs showed binding patterns that resemble the YxFW motif in the 10E8 HCDR3 loop. Additionally, we demonstrate that high-resolution maps can be generated from heterogeneous samples with pooled competing Fabs. Overall, our findings will facilitate the optimization of MPER GT-antigens and push the size limit for cryoEM-based epitope mapping with smaller antigens and heterogeneous antibody mixes. HighlightsO_LIElucidate the structural basis of increased 10E8 affinity to GT12 over GT10.2 C_LIO_LICryoEM epitope mapping reveals three main binding sites on MPER-GT immunogens C_LIO_LIProof of concept for application of CryoEM epitope mapping to small immunogens C_LIO_LIStructural characterization and MD analysis confirms 10E8-like interactions of the MPER-specific Fabs C_LI

immunology↗

bioRxiv2025

Virus glycoprotein nanodisc platform for vaccine design

Transmembrane glycoproteins of enveloped viruses are the targets of neutralizing antibodies and essential vaccine antigens. mRNA-LNP technology allows in situ production of transmembrane glycoproteins upon immunization, but biophysical characterization of transmembrane antigens and in vitro analysis of post-immunization antibody responses typically rely on soluble proteins. Here, we present a methodological platform for assembling transmembrane glycoprotein vaccine candidates into lipid nanodiscs. We demonstrate the utility of the nanodiscs in HIV membrane proximal external region (MPER)-targeting vaccine development by binding assays using surface plasmon resonance (SPR), ex vivo B cell sorting with fluorescence-activated cell sorting (FACS), and by determining the structure of a prototypical HIV MPER-targeting immunogen nanodisc in complex with three broadly neutralizing antibodies (bnAbs), including the MPER bnAb 10E8, to 3.5 [A] by cryogenic electron microscopy (cryo-EM), providing a template for structure-based immunogen design for MPER. Overall, the platform offers a tool for accelerating the development of next-generation viral vaccines.

immunology↗

bioRxiv2025

DNA origami vaccines program antigen-focused germinal centers

Recruitment and expansion of rare precursor B cells in germinal centers (GCs) is a central goal of vaccination to generate broadly neutralizing antibodies (bnAbs) against challenging pathogens such as HIV. Multivalent immunogen display is a well-established method to enhance vaccine-induced B cell responses, typically accomplished by using natural or engineered protein scaffolds. However, these scaffolds themselves are targets of antibody responses, with the potential to generate competitor scaffold-specific B cells that could theoretically limit expansion and maturation of "on-target" B cells in the GC response. Here, we rationally designed T-independent, DNA-origami based virus-like particles (VLPs) with optimal antigenic display of the germline targeting HIV Env immunogen, eOD-GT8, and appropriate T cell help to achieve a potent GC response. In preclinical mouse models, these DNA-VLPs expanded significantly higher frequencies of epitope-specific GC B cells compared with a state-of-the-art clinical protein nanoparticle. Optimized DNA-VLPs primed germinal centers focused on the target antigen and rapidly expanded subdominant broadly neutralizing antibody precursor B cells for HIV with a single immunization. Thus, avoiding scaffold-specific responses augments priming of bnAb precursor B cells, and DNA-VLPs are a promising platform for promoting B cell responses towards challenging subdominant epitopes.

immunology↗

bioRxiv2025

Vaccination with mRNA-encoded membrane-bound HIV Envelope trimer induces neutralizing antibodies in animal models

A protective vaccine against HIV will likely need to induce broadly neutralizing antibodies (bnAbs) that engage relatively conserved epitopes on the HIV envelope glycoprotein (Env) trimer. Nearly all vaccine strategies to induce bnAbs require the use of relatively complex immunization regimens involving a series of different immunogens, most of which are Env trimers. Producing protein-based clinical material to evaluate such relatively complex regimens in humans presents major challenges in cost and time. Furthermore, immunization with HIV trimers as soluble proteins induces strong non-neutralizing responses to the trimer base, which is normally occluded on the virion. These base responses could potentially detract from the induction of nAbs and the eventual induction of bnAbs. mRNA vaccine platforms offer potential advantages over protein delivery for HIV vaccine development, including increased production speed, reduced cost, and the ability to deliver membrane-bound trimers that might facilitate improved immuno-focusing to non-base epitopes. We report the design of mRNA-delivered soluble and membrane-bound forms of a stabilized native-like Env trimer (BG505 MD39.3), initial immunogenicity evaluation in rabbits that triggered clinical evaluation, and more comprehensive evaluation of B cell, T cell, and antibody responses in non-human primates. mRNA-encoded membrane-bound Env immunization elicited reduced off-target base-directed Env responses and stronger neutralizing antibody responses, compared with mRNA-encoded soluble Env. Overall, mRNA delivery of membrane-bound Env appears promising for enhancing B cell responses to subdominant epitopes and facilitating rapid translation to clinical testing, which should assist HIV vaccine development. One Sentence SummaryHIV envelope trimer mRNA enables membrane-bound expression and represents a functional immunogen in pre-clinical mammalian models.

immunology↗

bioRxiv2024

Diverse competitor B cell responses to a germline-targeting priming immunogen in human Ig loci transgenic mice

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.

immunology↗

bioRxiv2023

Engineered display of ganglioside-sugars on protein elicits a clonally and structurally constrained B cell response

Ganglioside sugars, as Tumour-Associated Carbohydrate Antigens (TACAs), are long-proposed targets for vaccination and therapeutic antibody production, but their self-like character imparts immunorecessive characteristics that classical vaccination approaches have to date failed to overcome. One prominent TACA, the glycan component of ganglioside GM3 (GM3g), is over-expressed on diverse tumours. To probe the limits of glycan tolerance, we used protein editing methods to display GM3g in systematically varied non-native presentation modes by attachment to carrier protein lysine sidechains using diverse chemical linkers. We report here that such presentation creates glycoconjugates that are strongly immunogenic in mice and elicit robust antigen-specific IgG responses specific to GM3g. Characterisation of this response by antigen-specific B cell cloning and phylogenetic and functional analyses suggests that such display enables the engagement of a highly restricted naive B cell class with a defined germline configuration dominated by members of the IGHV2 subgroup. Strikingly, structural analysis reveals that glycan features appear to be recognised primarily by antibody CDRH1/2, and despite the presence of an antigen-specific Th response and B cell somatic hypermutation, we found no evidence of affinity maturation towards the antigen. Together these findings suggest a reach-through model in which glycans, when displayed in non-self formats of sufficient distance from a conjugate backbone, may engage glycan ready V-region motifs encoded in the germline. Structural constraints define why, despite engaging the trisaccharide, antibodies do not bind natively-presented glycans, such as when linked to lipid GM3. Our findings provide an explanation for the long-standing difficulties in raising antibodies reactive with native TACAs, and provide a possible template for rational vaccine design against this and other TACA antigens. HighlightsO_LIGM3g synthetically coupled via a longer, orthogonal (from backbone) glycoconjugate (LOG) presentation format (thioethyl-lysyl-amidine) display elicits high-titre IgG responses in mice. C_LIO_LIThe germinal centre experience of LOG glycoconjugate-specific B cell responses is directly influenced by the protein backbone. C_LIO_LIStructural characterisation of the antibody response to LOGs reveals highly restricted germline-encoded glycan-engaging motifs that mediate GM3g recognition. C_LIO_LIFailure of antibodies to bind the native trisaccharide highlights barriers to be overcome for the rational design of anti-TACA antibodies. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/543556v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@b2111org.highwire.dtl.DTLVardef@a81b88org.highwire.dtl.DTLVardef@a78c6aorg.highwire.dtl.DTLVardef@1f3b4b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens

Chemical cross-linking is used to stabilise protein structure with additional benefits of pathogen and toxin inactivation for vaccine use, but its use is restricted by potential induction of local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for the purposes of understanding function, or for inducing a biological outcome such as elicitation of antibodies to conformationally-sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its functional analysis and use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent EDC to cross-link two soluble Env trimers, selected well-folded trimers using an antibody affinity column, and transferred this process to good manufacturing practice (GMP) for clinical trial use. Cross-linking enhanced GMP trimer stability to biophysical and enzyme attack, and had broadly beneficial effects on morphology, antigenicity and immunogenicity. Cryo-EM analysis revealed that cross-linking essentially completely retained overall structure with RMSDs between unmodified and cross-linked Env trimers of 0.4-0.5 [A]. Despite this negligible distortion of global trimer structure we identified individual inter-subunit, intra-subunit and intra-protomer cross-links. Thus, EDC cross-linking maintains protein folding, improves stability, and is readily transferred to GMP, consistent with use of this approach in probing protein structure/function relationships and in the design of vaccines.

molecular biology↗

bioRxiv2021

An ultrasensitive method for analysis of viral spike N-glycoforms

Viruses can evade the host immune system by displaying numerous glycans on their surface "spike-proteins" that cover immune epitopes. We have developed an ultrasensitive "single pot" method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for analysis of site-specific glycosylation of any glycoprotein.

biochemistry↗

bioRxiv2020

Vaccine Elicitation of HIV Broadly Neutralizing Antibodies from Engineered B cells

HIV broadly neutralizing antibodies (bnAbs) can suppress viremia and protect against infection1. However, their elicitation is made difficult by low frequencies of appropriate precursor B cell receptors and the complex maturation pathways required to generate bnAbs from these precursors2. Antibody genes can be engineered into B cells for expression as both a functional receptor on cell surfaces and as secreted antibody3-5. Here, we show that HIV bnAb-engineered primary mouse B cells can be adoptively transferred and vaccinated in immunocompetent wild-type animals resulting in the expansion of durable bnAb memory and long-lived plasma cells. Somatic hypermutation after immunization indicated that engineered cells have the capacity to respond to an evolving pathogen. These results encourage further exploration of engineered B cell vaccines as a strategy for durable elicitation of HIV bnAbs to protect against infection and as a contributor to a functional cure.

immunology↗

bioRxiv2020

Targeting HIV Env immunogens to B cell follicles in non-human primates through immune complex or protein nanoparticle formulations

Following immunization, high affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble HIV envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single lymph node within 2 days after immunization. Imaging of LNs collected 7 days post-immunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent germinal centers. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with immune complexes or nanoparticles.

immunology↗

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↗