Biology subjects

Toth, E. A.

Publications and source records attributed to Toth, E. A..

11 recordsLinked to original sources

bioRxiv2026

Incorporation of native HCV E1E2 into a nanoparticle vaccine platform

Development of an effective HCV vaccine requires the induction of both broadly neutralizing antibodies (bnAbs) and a robust cellular response. One issue that has arisen is that HCV subunit vaccines have limited immunogenicity, thus requiring multivalent formats in order to elicit a robust anti-HCV immune response. Toward that end, nanoparticle vaccines possess the ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both arms of the immune system. Here, we used a soluble, secreted form of E1E2 (sE1E2) to assemble native E1E2 into a nanoparticle platform using a post-purification coupling assembly system. Nanoparticles were assembled by purifying sE1E2 containing a C-terminal SpyTag and an mi3-SpyCatcher fusion separately and covalently coupling the components via incubation. Free sE1E2-SpyTag was removed from nanoparticle preparations via gel filtration. The sE1E2-mi3 nanoparticles are fully competent to bind conformation-dependent bnAbs, indicating retention of a native assembly in the nanoparticle format. Electron microscopy analysis showed a clear incorporation of sE1E2 on the surface of the nanoparticle. Immunogenicity of sE1E2-mi3 nanoparticles was examined relative to sE1E2 alone and membrane-bound E1E2 (mbE1E2) following inoculation of groups of CD1 mice. Assessment of the immunogenicity of the sE1E2-mi3 nanoparticles showed that the nanoparticle assembly has a similar immunogenicity profile to that of mbE1E2 after only a prime and one boost, and overall superior to sE1E2. This proof-of-principle study sets the stage for further exploration of nanoparticles and other multivalent platforms for the development of E1E2-based vaccines. ImportanceHepatitis C virus infects approximately 50 million people, and at present no effective HCV vaccine exists. Due to the high sequence variability of HCV and the resulting difficulty in developing a vaccine that elicits a broadly neutralizing response, multiple efforts are underway to enhance the immunogenicity of HCV vaccine candidates. In this study, we incorporated native soluble, secreted E1E2 (sE1E2) into a 60-mer nanoparticle via the SpyTag-SpyCatcher system and covalent isopeptide bond attachment using the purified components. These nanoparticles are antigenically intact and elicit a neutralizing antibody response at an earlier time point in the immunization regimen than the corresponding subunit vaccine. These studies show that a well-characterized sE1E2 platform compatible with multiple genotypes can be coupled to nanoparticles for use as a vaccine candidate.

microbiology↗

bioRxiv2026

Engineered subtilisin protease degrades active KRAS in cancer cells, leading to differential cell targeting

Controlling aberrant RAS signaling has been the subject of intensive efforts aimed at developing specific RAS inhibitors, small molecules that promote RAS degradation, and monobodies that inhibit RAS activity. Direct proteolytic degradation of RAS by site-specific proteases has received considerably less attention. A naturally-occurring protease from Vibrio vulnificus toxin cleaves all RAS isoforms at switch I and attenuates RAS signaling in cell models and patient-derived xenografts, thus demonstrating the potential of this approach. We previously designed a RAS-specific protease, called RASProtease (or RASp), that site-specifically cleaves RAS at switch II. Attacking switch II leverages an order to disorder transition that this region undertakes upon conversion to the active form that predominates in cancer. Switch II participates in an allosteric network that controls KRAS oncogenicity, making it a promising target for proteolytic cleavage that modulates RAS signaling. Preferential targeting of active RAS could be particularly useful for studying RAS signaling networks as well as having potential therapeutic value. Here we examined the effects of RASp cleavage on downstream signaling and cell viability in the MIA PaCa-2 cancer cell model, which harbors homozygous KRAS G12C and is KRAS-dependent for growth and survival. We found that cleavage of KRAS G12C coincided with a decrease in MEK-ERK signaling and resulted in extensive MIA PaCa-2 cell death 24 hours after induction of RASp expression. This level of cell death far exceeded that of control HEK 293T cells under the same conditions, underscoring the vulnerability of this cancer cell model to KRAS G12C elimination.

cancer biology↗

bioRxiv2026

Substrate specificity in a designed RAS-targeting protease is coupled to active site and distal motions

Designing proteases with tailored substrate specificity has emerged as a powerful strategy for manipulating protein function in cells. RAS, a key regulator of cell survival and proliferation, is a compelling target for such approaches. Mutations in RAS are involved in about one-third of all human cancers and drive the hyperactive signaling that promotes tumorigenesis, growth, and metastasis in cancers such as pancreatic and lung cancer. This creates a pressing need for strategies capable of modulating mutant RAS with high substrate specificity to avoid unintended cleavage events. As a model for targeted proteolysis, we present the high-resolution crystal structures of RASProtease(II), which provide a detailed view of the enzymes active site and substrate-binding architecture. Kinetic experiments showed that cleavage of the cognate QEEYSAM substrate is approximately 30-fold faster than the non-cognate QEEISAM, demonstrating strong proteolytic selectivity. NMR dynamics studies combined with structural mapping revealed that substrate binding modulates not only the active site, but also distal regions of RASProtease(II), uncovering long-range allosteric networks. Contrary to the conventional view that non-cognate substrates are simply poor fits for the active site, we found that binding of the non-cognate peptide induces a greater amount of conformational dynamics in the protease than in the apo form or cognate complex, resulting in significant destabilization and providing a mechanistic explanation for the reduced catalytic efficiency. These results reveal how distal structural networks help define substrate specificity and provide principles for rationally designing proteases with enhanced specificity for therapeutic applications. Significance StatementDesigner proteases are gaining interest for their potential as therapeutic agents. It is therefore critical to understand the mechanisms that differentiate cognate and non-cognate sequences to minimize deleterious off-target cleavage events. While the conventional view is that non-cognate substrates are simply poor fits for the active site, we demonstrate here that dynamic allostery plays an integral role in distinguishing between cognate and off-target sequences, using a protease designed to target the oncoprotein RAS as our model system. Moreover, because the RASProtease is derived from a prototypical serine protease, subtilisin, it is likely that the overall mechanism employed for selecting cognate over non-cognate substrates presented here will be generalizable, providing principles for designing enhanced specificity in a wide range of proteases.

biochemistry↗

bioRxiv2026

mAbClust with AlphaFold 3 avoids hallucinations to define a quaternary broadly neutralizing HCV epitope

The hepatitis C virus (HCV) envelope glycoprotein E1E2 heterodimer is the target of broadly neutralizing antibodies (bNAbs). Although prior studies have indicated that E1-dependent bNAbs are associated with spontaneous clearance of HCV, all E1-dependent human monoclonal antibodies (mAbs) have been isolated from individuals with chronic HCV infection. Here, we isolated E1-dependent bNAbs from an individual with high neutralizing antibody breadth who spontaneously cleared HCV, showing that these bNAbs bind to four distinct sites on E1E2. We also developed mAbClust, an algorithm that improves identification of accurate AlphaFold 3 (AF3) structure predictions of antigen-antibody complexes. We used AF3 and mAbClust to generate a high-confidence predicted structure of an E1-dependent bNAb in complex with E1E2, showing that this bNAb binds to a quaternary epitope spanning E1 and E2. This study identifies four neutralizing sites and a quaternary bNAb epitope associated with HCV control, which can guide HCV vaccine design. AF3 with mAbClust could have broad applications for accurate epitope mapping of antibodies.

immunology↗

bioRxiv2025

B cell transcriptomics reveals lasting dysregulation and rapid decline of protective memory after hepatitis C cure

O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/664545v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@d12426org.highwire.dtl.DTLVardef@9c54daorg.highwire.dtl.DTLVardef@186d559org.highwire.dtl.DTLVardef@120f2f3_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG HighlightsO_LIB cells in CHC patients retained dysregulated transcriptional profiles despite successful DAA treatment C_LIO_LIB cell dysregulation is marked by global B cell hyperactivation and antigen-specific atypical MBC expansion C_LIO_LISustained upregulation of TNF- signaling via NF-{kappa}B (TNF-/NF-{kappa}B) is a central driver of persistent B cell dysregulation and chronic inflammation C_LIO_LIViral clearance leads to restoration of IFN responses and IFN-stimulated gene (ISG) signatures in B cells but not TNF-/NF-{kappa}B C_LIO_LIHCV IGHV1-69 bnAb-producing B cells are enriched within the CD86hi IgG MBC subset before treatment C_LIO_LICD86hi IgG MBC subset contracts rapidly post-cure in parallel with the rapid decline of cross-nAb responses and loss of protective immune memory against HCV reinfection C_LI Chronic hepatitis C (CHC) disrupts host humoral immune response by impairing the timely generation of neutralizing antibody (nAb) and durable immune memory. However, the underlying mechanisms and their reversibility after viral clearance remain poorly defined. Here, through integrated single-cell transcriptomics and antibody repertoire characterization, we show that B cells from CHC patients retain transcriptional dysregulation even after successful antiviral therapy. Sustained TNF- signaling emerged as a central driver of chronic B cell hyperactivation, persistent dysregulation and unresolved inflammation following cure. Furthermore, a CD86hi memory B cell subset, responsible for an IGHV1-69-encoded multi-donor class recall nAb response, declined rapidly following viral clearance, compromising immune memory against reinfection. Together, these findings reveal how CHC imprints lasting B cell dysregulation, impairs nAb memory, and sustains inflammation in the B cell compartment, after viral clearance. The insights underscore the need for strategies aimed at restoring B cell homeostasis to achieve durable immune protection.

immunology↗

bioRxiv2025

Enhanced Production of HCV E1E2 Subunit Vaccine Candidates via Protein-Protein Interaction Identification in Glycoengineered CHO cells

Hepatitis C Virus (HCV) is a bloodborne virus that affects 57 million people globally with infections that can often go unnoticed, and it is the leading cause of chronic liver disease and cancer. Thus, development of an HCV vaccine is a major medical and public health concern. While prior work has developed secreted E1E2 (sE1E2) protein vaccine candidates, efforts to express it recombinantly in Chinese hamster ovary (CHO) cells have resulted in very low titers. To address this challenge, here we employed a multi-omics approach to identify protein interactors that enhance the secretion of sE1E2. By detecting Protein-Protein Interactions (PPIs) using Biotinylation by Antibody Recognition (BAR) and integrating the data with RNA-Seq, we identified proteins within the secretory pathway that interact with sE1E2 and validated their impact by overexpressing the interacting proteins. Among these, CUL4A and YWHAH enhanced sE1E2 secretion in glycoengineered CHO (geCHO) cells. The integration of omics techniques and genetic engineering in this study provides valuable insights into improving protein secretion in CHO cells, paving the way for the development of more affordable and accessible biotherapeutics.

systems biology↗

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

Glycoengineering of the hepatitis C virus E2 glycoprotein leads to improved biochemical properties and enhanced immunogenicity

An effective vaccine against hepatitis C virus (HCV) must elicit the production of broadly neutralizing antibodies (bnAbs) reproducibly against the E1E2 glycoprotein complex. Little is known about how glycan content affects this process. Ideally, glycans would maximize epitope exposure without compromising antigen stability or exposing new epitopes. However, typical recombinant vaccines contain considerable heterogeneity in glycan content, which can affect the antibody response and neutralization potency. Here we employed glycoengineered Chinese hamster ovary (geCHO) cell lines that impart nearly homogeneous glycosylation as a means to test how specific glycan features influence antigenicity and immunogenicity for the secreted HCV E2 ectodomain (sE2). Specific geCHO antigens exhibited a modest but reproducible increase in affinity for some mAbs relative to CHO- and HEK293-produced sE2. Surprisingly, one geCHO sE2 antigen failed to bind the CD81 receptor, indicating the potential for significant glycan effects on biochemical properties. We immunized mice with the four antigens and found the total antibody response to be the same for all groups. However, sera from one geCHO group exhibited a 7-fold improvement in neutralization against the homologous HCV pseudovirus and had the most mice whose sera exhibited neutralization activity against genotypes 1b, 2a, 2b, and 3. Further analysis identified beneficial and deleterious glycan features, and the glycan that correlated the most with decreased potency was relatively small. However, size was not the sole determinant of glycan-driven effects on the antibody response. In summary, glycan content impacts biochemical properties of antigens to varying degrees and such effects can influence immune response quality and uniformity.

immunology↗

bioRxiv2024

Glycoengineered recombinant alpha1-antitrypsin results in comparable in vitro and in vivo activities to human plasma-derived protein

Alpha-1-antitrypsin (A1AT) is a multifunctional, clinically important, high value therapeutic glycoprotein that can be used for the treatment of many diseases such as alpha-1-antitrypsin deficiency, diabetes, graft-versus-host-disease, cystic fibrosis and various viral infections. Currently, the only FDA-approved treatment for A1AT disorders is intravenous augmentation therapy with human plasma-derived A1AT. In addition to its limited supply, this approach poses a risk of infection transmission, since it uses therapeutic A1AT harvested from donors. To address these issues, we sought to generate recombinant human A1AT (rhA1AT) that is chemically and biologically indistinguishable from its plasma-derived counterpart using glycoengineered Chinese Hamster Ovary (geCHO-L) cells. By deleting nine key genes that are part of the CHO glycosylation machinery and expressing the human ST6GAL1 and A1AT genes, we obtained stable, high producing geCHO-L lines that produced rhA1AT having an identical glycoprofile to plasma-derived A1AT (pdA1AT). Additionally, the rhA1AT demonstrated in vitro activity and in vivo half-life comparable to commercial pdA1AT. Thus, we anticipate that this platform will help produce human-like recombinant plasma proteins, thereby providing a more sustainable and reliable source of therapeutics that are cost-effective and better-controlled with regard to purity, clinical safety and quality.

bioengineering↗

bioRxiv2020

Engineering protein-specific proteases: targeting active RAS

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

biochemistry↗

bioRxiv2020

Structure-based design of hepatitis C virus E2 glycoprotein improves serum binding and cross-neutralization

An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of bNAbs in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV.

microbiology↗