Biology subjects

Klein, F.

Publications and source records attributed to Klein, F..

At least 19 recordsLinked to original sources

bioRxiv2026

B cell response to HCMV gH/UL116 elicits neutralizing and FcγR-activating antibodies

Humoral immune mechanisms that can contribute to limiting HCMV disease include neutralizing antibodies that block viral entry and Fc-mediated effector functions that promote elimination of infected cells. Here, we identify the gH/UL116 envelope complex as a naturally immunogenic target that elicits antibodies capable of mediating both of these immune mechanisms. Using single-cell sorting of antigen-specific memory B cells, we isolated gH/UL116-reactive monoclonal antibodies from HCMV-seropositive donors. Epitope mapping showed that gH binding was required for virus neutralization, whereas UL116 targeting was associated with potent and broad Fc{gamma} receptor (Fc{gamma}R) activation. Furthermore, cryo-electron microscopy provided high-resolution structures revealing how UL116-specific antibodies bind gH/UL116. In mice, gH/UL116 immunization elicited serum IgG responses whose binding titers strongly correlated with both neutralization and mouse Fc{gamma}R activation. Together, these findings provide an epitope-level map linking antibody specificity to neutralization and Fc{gamma}R activation across HCMV gH-associated complexes and position gH/UL116 as a promising HCMV vaccine candidate.

immunology↗

bioRxiv2026

Durable suppression of viremia by lipid nanoparticle-formulated RNA encoding for a highly potent HIV-1 neutralizing antibody

Recombinant broadly neutralizing antibodies (bNAbs) are promising tools to treat and prevent HIV-1 infection but are associated with manufacturing challenges. To overcome this limitation, we encoded the potent bNAb 1-18-LS on RNA (1-18-LS RibobNAb) for delivery via lipid nanoparticles (LNPs). Indicating high in vivo antibody expression, a 30 ug RNA-LNP intravenous injection resulted in an average peak 1-18-LS RibobNAb serum concentration of 1,061 ug/mL in human neonatal Fc receptor-transgenic mice. Notably, weekly 30 ug RNA-LNP doses maintained higher trough bNAb levels than 500 ug protein injections. Highlighting potent antiviral activity, RNA-LNP-mediated 1-18-LS RibobNAb monotherapy of viremic HIV-1YU2-infected humanized mice resulted in durable HIV-1 suppression without emerging viral escape. Importantly, 1-18-LS RNA-LNP treatment also fully controlled infection after interruption of antiretroviral therapy in humanized mice infected with different patient-derived polyclonal HIV-1 isolates. Our findings provide a proof-of-principle for effective RNA-mediated bNAb immunotherapy of HIV-1 infection.

immunology↗

bioRxiv2026

Identification of a pan-orthoebolavirus-reactive antibody from an rVSV-EBOV vaccinated individual

Orthoebolaviruses such as Ebola virus (EBOV), Sudan virus (SUDV) and Bundibugyo virus (BDBV) can cause severe disease with high case-fatality rates. While licensed EBOV vaccines and therapeutic antibodies protect against EBOV infection, no single monoclonal antibody currently provides broad protection across multiple orthoebolaviruses. Here, we analyzed the humoral immune response of an rVSV-EBOV vaccinee to identify pan-orthoebolavirus-neutralizing antibodies. Using BDBV- and SUDV-glycoproteins for single B cell-sorting, we identified B10, which neutralized authentic EBOV and SUDV, with potent activity against SUDV compared with established cross-reactive antibodies. Structural analysis mapped antibody B10 binding to the pan-orthoebolavirus conserved GP2-stalk/HR2 region, associated with asymmetric trimer destabilization and spike opening. In vivo, B10 showed significant prophylactic efficacy in an EBOV mouse model and partial protection with antiviral activity in a SUDV mouse model. Together, these findings demonstrate that rVSV-EBOV vaccination induced the development of a broadly orthoebolavirus-neutralizing antibody that holds exeptional therapeutic potential.

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

Structural characterization of antibodies for synergistic HCMV neutralization

The limited mechanistic understanding of Human Cytomegalovirus (HCMV) infection and neutralization has hindered the effective application of antibodies for HCMV prevention and therapy. To address this, we conducted high-resolution cryo-EM analysis of 17 human monoclonal antibodies targeting HCMV glycoproteins gH and gL. This analysis revealed a landscape of vulnerable epitopes, including detailed binding modes, spatial orientations, and structural features associated with distinct neutralization phenotypes. By comprehensively characterizing the neutralizing properties of these antibodies and their combinations, we identified synergistic antibody cocktails that enhance the neutralization capacity of individual antibodies. Notably, while single antibodies only partially neutralized HCMV, their synergistic combinations achieved substantially more complete inhibition. Together, these findings delineate structural correlates of HCMV neutralization and establish functional principles for antibody-based combinatorial targeting of HCMV.

immunology↗

bioRxiv2026

Memory T Cells in MHC-Deficient Humanized Mice

Major histocompatibility complexes (MHC) govern antigen presentation and T cell receptor (TCR) selection. Accurate in vivo modeling of human immunity therefore requires physiological human MHC TCR interactions. Humanized NOD scid IL2null (NSG) mice engrafted with human CD34+ hematopoietic stem cells are widely used to provide preclinical platforms for the development of advanced therapies; however, interactions between murine MHC and human TCR can promote xenoreactivity and alter T cell development. Here, we investigated how elimination of murine MHC together with different conditioning regimens shapes human T cell maturation in vivo. CD34+ cells from ten cord blood donors were transplanted into conventional NSG mice or murine MHC deficient NSG derivatives (DKO) following either sublethal irradiation or myeloablative busulfan conditioning. Integrated analyses combining flow cytometry, plasma cytokine profiling, and bulk and single cell TCR sequencing revealed marked differences in T cell differentiation across models. Busulfan conditioned DKO mice developed highly proliferative, activated, and cytotoxic T cells together with clonally expanded TCR repertoires. In contrast, irradiated NSG mice preferentially accumulated naive, NKT, and regulatory T cell populations. Busulfan-conditioned DKO mice showed no evidence of xenogeneic graft versus host disease and represent a refined enabling platform for human T cell development and provide a foundation for future preclinical evaluation of advanced gene and cell therapies.

immunology↗

bioRxiv2026

The TREM2 targeting small molecule Sob-AM2 improves cognition independent of Aβ plaque alteration in 5xFAD mice

BackgroundInflammation is an early event that substantially influences Alzheimers disease (AD) pathogenesis, making it a compelling target for therapeutic intervention. Sob-AM2 is a brain-penetrating thyromimetic drug capable of inducing the expression of microglial cell surface receptor TREM2, which mediates the switch from pro-inflammatory to a more restorative microglial state. ObjectiveEvaluate the effects of Sob-AM2 on cognition, AD pathology and microglial activity in the 5xFAD mouse model of amyloid-beta (A{beta}) accumulation. MethodsSeven-month-old 5xFAD mice and their wild-type littermates were administered Sob-AM2 subcutaneously three times per week for 12 weeks. In the last two weeks of treatment mice underwent behavioral tests to assess cognition and monitor for off-target mobility effects. At the end of treatment, brain tissue was harvested for gene and protein expression analyses. ResultsSob-AM2 treatment increased TREM2 expression in the brains of 5xFAD mice. This was accompanied by an improvement in both spatial and associative memory as well as an increase in the expression of synaptic genes synaptophysin and PSD-95. No significant changes were detected in A{beta} plaque burden or the expression of microglial activation marker Iba1 in Sob-AM2 treated animals, however, the expression of the phagocytic marker CD68 was significantly increased in the hippocampus, but not the cortex, in Sob-AM2 treated 5xFAD mice. ConclusionThese results suggest that the cognitive-enhancing effects of Sob-AM2 are not the result of reduced overall plaque burden. Future work is needed to further investigate the neuroprotective mechanism of Sob-AM2 and how it may be affecting microglial phenotypes.

neuroscience↗

bioRxiv2026

Microbial Growth in an Enceladus Ocean Analog Medium Informed by Mineral Stability Modeling

Evidence from the Cassini mission confirmed that Saturns moon Enceladus hosts a subsurface alkaline ocean where rock-water reactions may generate redox disequilibria capable of supporting microbial metabolisms. To investigate potential microbial survival under simulated Enceladus ocean conditions, we used thermodynamic modeling to develop a salt formulation consistent with one possible Enceladus ocean composition and supplemented it with putative microbial energy sources to create a growth medium. The medium was inoculated with samples from diverse ocean world analog environments on Earth to determine which microorganisms could persist under Enceladus-like conditions. The microorganisms persisting in this geochemically bounded medium were heterotrophic, metabolically versatile bacteria with low carbon requirements. Genomic and physiological analyses further showed the presence of multiple stress-response pathways, sodium-based bioenergetic systems, osmoregulation strategies, and other adaptations consistent with survival in alkaline, low-nutrient settings. These results suggest that some stress-tolerant heterotrophic bacteria may serve as useful model organisms for life in Enceladus subsurface ocean. These findings demonstrate the value of geochemically modeled media as a framework for constraining habitability, identifying relevant biosignatures, and probing potential microbial survival strategies beyond Earth.

microbiology↗

bioRxiv2025

A platform of robust patient-derived leukemia models covering subgroups for which no cell lines exist

Preclinical cancer research requires robust model systems, especially for poor prognosis entities like acute myeloid leukemia (AML), a highly aggressive blood cancer. Here, primary tumor cells from 137 AML patients of all age groups were transplanted into immune compromised mice to generate patient-derived xenografts (PDX). From these, 23 models enable robust, virtually endless serial re-transplantation and are amenable to lentiviral genetic engineering (*PDX AML models). These models primarily originate from patients with highly aggressive, relapsed disease. Comprehensive genomic, transcriptomic, and epigenomic analyses confirmed that they replicate primary AML biology more faithfully than conventional cell lines. Notably, *PDX AML models include AML subgroups that are underrepresented or absent in existing model systems, such as cytogenetically normal or IDH1/2-mutant AML. They withstand freeze-thaw cycles, making them suitable for broad distribution and reproducibility across research institutions. Luciferase-based in vivo imaging enables real-time monitoring of tumor progression and treatment responses in preclinical trials. Surprisingly, long-term treatment, including repeated cytarabine therapy over a period of one year, showed a gradual reduction in leukemia cell proliferation, which decreased continuously after each treatment block. Collectively, our *PDX models represent a robust, versatile, and relevant platform that holds great promise to accelerate translational research for the benefit of cancer patients. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/677299v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1ead31eorg.highwire.dtl.DTLVardef@1ca702corg.highwire.dtl.DTLVardef@18a7137org.highwire.dtl.DTLVardef@edbe6_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIWe present new robust AML PDX models covering subgroups for which no cell lines exist for use in various ex vivo and in vivo applications. C_LIO_LI*PDX models enable serial transplantation, genetic engineering and better representation of primary AML biology than cell lines. C_LIO_LIOne-year in vivo trials mimicking clinical chemotherapy showed surprising gradual decline in leukemia growth after each treatment block. C_LI

cancer biology↗

bioRxiv2025

Identification of a broad and potent V3 glycan site bNAb targeting anN332gp120 glycan-independent epitope

Broadly neutralizing antibodies (bNAbs) against HIV-1 can suppress viremia in vivo and inform vaccine development. Here, we characterized 007, a V3 glycan site bNAb exhibiting high levels of antiviral activity against multiclade pseudovirus panels1-3 (GeoMean IC50 = 0.012 {micro}g/mL, breadth = 69%, 217 virus strains) by targeting a N332gp120 glycan-independent V3 epitope, a site of Env vulnerability to which only weakly neutralizing antibodies had previously been identified. Functional analyses demonstrated distinct binding and neutralization profiles compared to classical V3 glycan site bNAbs. A 007 Fab-Env cryo-EM structure revealed contacts with the V3 324GD/NIR327 motif and interactions with N156gp120 and N301gp120 glycans. In contrast to classical V3 bNAbs, 007 binding to Env does not depend on the N332gp120 glycan, rendering it resistant to common escape mutations. Structures of 007 IgG-Env trimer complexes showed two Env trimers crosslinked by three bivalent IgGs, and bivalent 007 IgG was up to [~]300-fold more potent than monovalent 007 IgG heterodimer, suggesting a role for avidity in potent neutralization. Finally, in HIV-1ADA-infected humanized mice, 007 caused transient decline of viremia and overcame classical V3 escape mutations, highlighting 007s potential for HIV-1 prevention, therapy, functional cure, and vaccine design.

immunology↗

bioRxiv2025

Detection of low pre-existing humoral immunity against influenza virus H5N1 clade 2.3.4.4b in unexposed individuals

The spill-over of Influenza A virus H5N1 clade 2.3.4.4b from cattle to humans highlights the risk of a human H5N1 pandemic. Given the impact of pre-existing immunity on the course and severity of viral infections, we comprehensively assessed the humoral immunity against the H5N1 A/Texas/37/2024 isolate in H5N1-naive individuals. To this end, we performed complementary binding and neutralization assays on 66 subjects and ranked activities among a panel of 76 influenza A virus isolates. We detected low but distinct cross-neutralizing titers against A/Texas/37/2024 with a 3.9 to 15.6-fold reduction compared to selected H1N1 or H3N2 strains. By cloning and evaluating 136 monoclonal antibodies from memory B cells, we identified potent A/Texas/37/2024-neutralizing monoclonal antibodies in five out of six investigated individuals. These antibodies cross-neutralize H1, compete with antibodies targeting the HA stem, and protect mice from lethal H5N1 challenge. Our findings demonstrate partial pre-existing humoral immunity to A/Texas/37/2024 in H5N1-naive individuals.

immunology↗

bioRxiv2025

Human cytomegalovirus gH/gL/gO binding to PDGFRα provides a regulatory signal activating the fusion protein gB that can be blocked by neutralizing antibodies.

Herpesviruses require membrane fusion for entry and spread, a process facilitated by the fusion glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL can be modified by the accessory protein gO, or the set of proteins UL128, UL130 and UL131. While the binding of the gH/gL/gO and gH/gL/UL128-131 complexes to cellular receptors including PDFGR and NRP2 has been well-characterized structurally, the specific role of receptor engagements by the gH/gL/gO and gH/gL/UL128-131 in regulation of fusion has remained unclear. We describe a cell-cell fusion assay that can quantitatively measure fusion on a timescale of minutes and demonstrate that binding of gH/gL/gO to PDGFR dramatically enhances gB-mediated cell-cell fusion. In contrast, gH/gL/pUL128-131-regulated fusion is significantly slower and gH/gL alone cannot promote gB fusion activity within this timescale. The genetic diversity of gO influenced the observed cell-cell fusion rates, correlating with previously reported effects on HCMV infectivity. Mutations in gL that had no effect on formation of gH/gL/gO or binding to PDGFR dramatically reduced the cell-cell fusion rate, suggesting that gL plays a critical role in linking the gH/gL/gO-PDGFR receptor-binding to activation of gB. Several neutralizing human monoclonal antibodies were found to potently block gH/gL/gO-PDGFR regulated cell-cell fusion, suggesting this mechanism as a therapeutic target. SIGNIFICANCEDevelopment of vaccines and therapeutics targeting the fusion apparatus of HCMV has been limited by the lack an in vitro cell-cell fusion assay that faithfully models the receptor-dependent fusion characteristic of HCMV entry. The cell-cell fusion assay described here demonstrated that the binding of gH/gL/gO to its receptor, PDGFR serves to regulate the activity of the fusion protein gB. Moreover, this regulatory mechanism is specifically vulnerable to inhibition by neutralizing antibodies. The cell-cell fusion assay described here provides a new tool to characterize neutralizing mAbs as therapeutic agents.

microbiology↗

bioRxiv2024

Aberrant prefrontal activity and arousal level correlate with action initiation and response vigor

Orchestrating learned Stimulus-Response (S-R) mappings has been suggested as one of the central functions of the prefrontal cortex (PFC). While S-R selective activity has been demonstrated, it remains unclear whether the strength of such activity is related to the vigor of the subsequent response. Here, we trained male rats to perform a Go/NoGo response task while head-fixed on a treadmill. This allowed us to record PFC (cingulate, area 24) single unit spiking, as well as running speed as a proxy for response vigor. We show that aberrant activation of the "wrong" S-R mapping is correlated with initiation of the incorrect response. The vigor of the incorrect response was directly related to the strength of the aberrant stimulus-evoked activity. A similar relationship was observed for pre-trial arousal state and response vigor. Our findings confirm the long-standing concept, established in psychology and cognitive neuroscience, that S-R mappings are directly related to response vigor. Moreover, we provide evidence for the often suggested but rarely tested relationship between arousal and response vigor and a potential underlying neuronal mechanism involving neuromodulation of S-R mapping activity. Significance statementThe concept of stimulus-response (S-R) mappings is fundamental in psychology and has been widely documented as a key function of the prefrontal cortex. Here, the authors directly link prefrontal single neuron mapping-selective activity to the vigor of responses. Moreover, they link a physiological measure of arousal to response vigor and suggest that neuromodulatory systems invigorate responses by potentially modulating PFC S-R mapping-selective activity.

neuroscience↗

bioRxiv2024

Temporal and Functional Relationship between Synaptonemal Complex Morphogenesis and Recombination during Meiosis

During prophase of meiosis I, programmed double strand breaks (DSBs) are processed into crossovers, a critical requirement for segregation of homologous chromosomes (homologs) and genome haploidization in sexually reproducing organisms. Crossovers form via homologous recombination in close temporospatial association with morphogenesis of the synaptonemal complex (SC), a proteinaceous structure that connects paired homologs along their length during the pachytene stage. Synapsis and recombination are a paradigm for the interplay between higher order chromosome structure and DNA metabolism, yet their temporal and functional relationship remains poorly understood. Probing linkage between these processes in budding yeast, we show that SC assembly is associated with a distinct threshold number of unstable D-loops. The transition from bona fide paranemic D-loops to plectonemic DSB single end invasions (SEIs) is completed during midpachynema, when the SC is fully assembled. Double Holliday junctions (dHJs) form at the time of desynapsis and are resolved into crossovers during diplonema. The SC central element component Zip1 shepherds recombination through three transitions, including DSB first end strand exchange and second end capture, as well as dHJ resolution. Zip1 mediates SEI formation independent of its polymerization whereas precocious Zip1 assembly interferes with double Holliday junction resolution. Together, our findings indicate that the synaptonemal complex controls recombination while assembled but also beyond its disassembly, possibly by establishing spatial constraints at recombination sites.

genetics↗

bioRxiv2023

Dissecting the impact of somatic hypermutation on SARS-CoV-2 neutralization and viral escape

Somatic hypermutation (SHM) drives affinity maturation and continues over months in SARS-CoV-2 neutralizing antibodies. Yet, several potent SARS-CoV-2 antibodies carry no or only few mutations, leaving the question of how ongoing SHM affects neutralization. Here, we reverted variable region mutations of 92 antibodies and tested their impact on SARS-CoV-2 binding and neutralization. Reverting higher numbers of mutations correlated with decreasing antibody functionality. However, some antibodies, including the public clonotype VH1-58, remained unaffected for Wu01 activity. Moreover, while mutations were dispensable for Wu01-induced VH1-58 antibodies to neutralize Alpha, Beta, and Delta variants, they were critical to neutralize Omicron BA.1/BA.2. Notably, we exploited this knowledge to convert the clinical antibody tixagevimab into a BA.1/BA.2-neutralizer. These findings substantially broaden our understanding of SHM as a mechanism that not only improves antibody responses during affinity maturation, but also counteracts antigenic imprinting through antibody diversification and thus increases the chances of neutralizing viral escape variants.

immunology↗

bioRxiv2023

Mapping the neutralizing specificity of human anti-HIV serum by deep mutational scanning

Understanding the specificities of human serum antibodies that broadly neutralize HIV can inform prevention and treatment strategies. Here we describe a deep mutational scanning system that can measure the effects of combinations of mutations to HIV envelope (Env) on neutralization by antibodies and polyclonal serum. We first show that this system can accurately map how all functionally tolerated mutations to Env affect neutralization by monoclonal antibodies. We then comprehensively map Env mutations that affect neutralization by a set of human polyclonal sera known to target the CD4-binding site that neutralize diverse strains of HIV. The neutralizing activities of these sera target different epitopes, with most sera having specificities reminiscent of individual characterized monoclonal antibodies, but one sera targeting two epitopes within the CD4 binding site. Mapping the specificity of the neutralizing activity in polyclonal human serum will aid in assessing anti-HIV immune responses to inform prevention strategies.

immunology↗

bioRxiv2022

Combined multidimensional single-cell protein and RNA profiling dissects the cellular and functional heterogeneity of thymic epithelial cells

The network of thymic stromal cells provides essential niches with unique molecular cues controlling T-cell development and selection. Recent single-cell RNA-sequencing studies uncovered a large transcriptional heterogeneity among thymic epithelial cells (TEC) demonstrating a previously unappreciated complexity. However, there are only very few cell markers that allow a comparable phenotypic identification of TEC. Here we deconvoluted by massively parallel flow cytometry and machine learning known and novel TEC phenotypes into novel subpopulations and related these by CITEseq to the corresponding TEC subtypes defined by the cells individual RNA profiles. This approach phenotypically identified perinatal cTEC, physically located these cells within the cortical stromal scaffold, displayed their dynamic change during the life course and revealed their exceptional efficiency in positively selecting immature thymocytes. Collectively, we have identified novel markers that allow for an unprecedented dissection of the thymus stromal complexity, the cells physical isolation and assignment of specific functions to individual TEC subpopulations.

immunology↗

bioRxiv2022

Neutralization sensitivity of the SARS-CoV-2 Omicron BA.2.75 sublineage

The recently emerged BA.2.75 Omicron sublineage of SARS-CoV-2 identified in numerous countries is rapidly increasing in prevalence in regions of India. Compared with BA.2, the spike protein of BA.2.75 differs in nine amino acid residues. To determine the impact of the spike mutations on polyclonal and monoclonal antibody activity, we investigated the neutralization sensitivity of BA.2.75 in comparison with B.1, BA.2, BA.2.12.1, and BA.4/5. Analysis of post-boost samples from 30 vaccinated individuals revealed significantly lower serum neutralizing activity against BA.2.75 than against BA.2. However, BA.2.75 was more sensitive to serum neutralization than the widely circulating BA.4/5 sublineages. Moreover, evaluation of 17 clinical-stage monoclonal antibodies demonstrated individual differences in Omicron sublineage activity. Notably, some authorized antibodies with low activity against other Omicron sublineages demonstrated high BA.2.75 neutralizing potency. Our results indicate a less pronounced degree of antibody evasion of BA.2.75 compared with BA.4/5 and suggest that factors beyond immune evasion may be required for an expansion of BA.2.75 over BA.4/5.

immunology↗