The induction of potent and cross-reactive neutralizing antibody (nAb) responses remains a challenge in vaccine development against antigenically diverse viruses such as hepatitis C virus (HCV). The HCV E1E2 glycoprotein complex contains two major neutralizing sites: the neutralizing face (NF) and the less explored bridging domain (BD). Here, we characterized 25 BD-targeting nAbs isolated from infection or immunization. These antibodies arise from diverse B cell lineages but share convergent CDRH3 features. Epitope mapping by alanine scanning and negative-stain electron microscopy revealed overlapping epitopes on BD spanning antigenic regions AR4 and AR5, with variable back layer engagement. The crystal structure of a non-human primate BD nAb RM3-26 in complex with E2 uncovered a back layer-directed recognition mode analogous to that of the human nAb hcab40. Together, BD- and NF-directed nAbs exhibited additivity in their neutralization, highlighting BD as a conserved site of vulnerability on HCV and a valuable target for rational vaccine design.

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 enzyme's 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.

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 A1AT deficiency, diabetes, graft-versus-host disease, cystic fibrosis, and various viral infections. Currently, the only U.S. food and drug administration-approved treatment for A1AT disorders is intravenous augmentation therapy with human plasma-derived A1AT (pdA1AT). 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 comparable to its plasma-derived counterpart using glycoengineered Chinese Hamster Ovary (geCHO-L) cells. By perturbing 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 a highly similar glycoprofile to 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 regarding purity, clinical safety, and quality.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. Development of an E1E2-based HCV vaccine has been hindered by the difficulty of producing a soluble E1E2 (sE1E2) antigen that faithfully recapitulates the native virion-associated heterodimer. Guided by cryo-electron microscopy (cryo-EM) structures, we engineer genotype 1a H77 sE1E2 by truncating the E1 and E2 stems (Cut1), deleting a putative fusion peptide-containing region in E1 (Cut2), and stabilizing the heterodimer using diverse scaffolds. All H77 sE1E2.Cut1+2 scaffolds exhibit native-like E1-E2 association and strong binding to the broadly neutralizing antibody (bNAb) AR4A. A genotype 1a HCV-1 sE1E2.Cut1+2 variant scaffolded by a modified SpyTag/SpyCatcher (SPYΔN) is selected for in vitro and in vivo characterization, as well as further construct refinement. The structure of this HCV-1 sE1E2 construct in complex with bNAbs is determined by cryo-EM and negative-stain EM (nsEM), with an nsEM-based strategy established for antibody epitope mapping. HCV-1 sE1E2.Cut1+2.SPYΔN is displayed on self-assembling protein nanoparticles (SApNPs) to enhance immunogenicity. The HCV-1 sE1E2.Cut1+2.SPYΔN heterodimer and SApNPs bearing wildtype or modified glycans are evaluated in mice, alongside E2 core-based immunogens for comparison. Together, these results establish a framework for advancing E1E2-based HCV vaccines toward clinical development.

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.

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. Importance:Hepatitis 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.

Background: The quest for well-defined immunoadjuvants remains one of the highest priorities for the successful development of effective vaccines. Combination adjuvants, which are designed to integrate both the ability to activate a variety of immune mechanisms and synergistically improve the delivery of vaccine components, are well-positioned to address the unmet needs. The development of a preventive vaccine against hepatitis C virus (HCV)—a major public health concern—is a particular instance in which the choice of the immunoadjuvant is of utmost importance. Methods: We assembled a lipid A Toll-like receptor 4 (TLR4) agonist BECC438 and TLR7/8 agonist resiquimod (R848) on a polyphosphazene macromolecule (PCPP) to create a nanoscale immunoadjuvant-vaccine delivery system: PCPP-R+BECC438. This aqueous-based system was formulated with the HCV sE2 antigen, and the resulting vaccine candidate was evaluated in vivo for the ability to induce immune responses. Results: Co-assembly of adjuvants resulted in a visually clear aqueous system of nanoscale dimensions, monomodal size distribution, and entropy-driven interactions between components. Intramuscular immunization of mice with HCV sE2 antigen formulated in a polyphosphazene-based nano-system induced ten-fold higher IgG and IgG2a titers than the antigen adjuvanted with BECC438 alone. PCPP-R+BECC438 formulated HCV sE2 also produced statistically significant improvements in IgG2a/IgG1 ratio and more robust HCVpp neutralization ID50 titers than control formulations. Conclusions: Polyphosphazene-assembled adjuvant nano-system promotes in vivo immune responses of enhanced quantity and quality of antibodies with increased potency of HCV neutralization.

Hepatitis C virus (HCV) is a major public health concern, and the development of an effective HCV vaccine plays an important role in the effort to prevent new infections. Supramolecular co-assembly and co-presentation of the HCV envelope E1E2 heterodimer complex and core protein presents an attractive vaccine design strategy for achieving effective humoral and cellular immunity. With this objective, the two antigens were non-covalently assembled with an immunostimulant (TLR 7/8 agonist) into virus-mimicking polymer nanocomplexes (VMPNs) using a biodegradable synthetic polyphosphazene delivery vehicle. The resulting assemblies were characterized using dynamic light scattering and asymmetric flow field-flow fractionation methods and directly visualized in their vitrified state by cryogenic electron microscopy. The in vivo superiority of VMPNs over the individual components and an Alum-formulated vaccine manifests in higher neutralizing antibody titers, the promotion of a balanced IgG response, and the induction of a cellular immunity—CD4+ T cell responses to core proteins. The aqueous-based spontaneous co-assembly of antigens and immunopotentiating molecules enabled by a synthetic biodegradable carrier offers a simple and effective pathway to the development of polymer-based supramolecular nanovaccine systems.

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.

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 (HCVpp) 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.

Hepatitis C Virus (HCV) is a pervasive bloodborne virus and the leading cause of chronic liver disease and cancer. Thus, the development of an HCV vaccine is of great importance. Prior work has developed candidate vaccines, including more potent glycoengineered viral proteins and secreted forms of the E1E2 envelope heterodimer (sE1E2). However, efforts to express them 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 may enhance the secretion of an sE1E2 vaccine candidate. We detected protein-protein interactions (PPIs) using the Biotinylation by Antibody Recognition (BAR) assay and integrated these data with RNA-Seq. Through this, we identified and overexpressed proteins that interact with sE1E2 in CHO cells. Among these, CUL4A and YWHAH enhanced sE1E2 secretion in our glycoengineered CHO cells. The integration of omics techniques and genetic engineering in this study provides valuable insights into the host cell proteins that interact with the HCV E1E2 heterodimer, and how they may be harnessed to improve protein secretion in CHO cells to enable more affordable and accessible biotherapeutics.

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-α 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.

Global elimination of hepatitis C virus (HCV) will require an effective cross-genotype vaccine. The HCV E2 envelope glycoprotein is the main target of neutralizing antibodies but also contains epitopes that elicit non-neutralizing antibodies which may provide protection through Fc effector functions rather than direct neutralization. We determined cryo-EM structures of a broadly neutralizing antibody, a moderately neutralizing antibody, and a non-neutralizing antibody bound to E2 to resolutions of 3.8, 3.3, and 3.7 Å, respectively. Whereas the broadly neutralizing antibody targeted the front layer of E2 and the non-neutralizing antibody targeted the back layer, the moderately neutralizing antibody straddled both front and back layers, and thereby defined a new neutralizing epitope on E2. The small size of complexes between conventional (monovalent) Fabs and E2 (~110 kDa) presented a challenge for cryo-EM. Accordingly, we engineered bivalent versions of E2-specific Fabs that doubled the size of Fab-E2 complexes and conferred highly identifiable shapes to the complexes that facilitated particle selection and orientation for image processing. This study validates bivalent Fabs as new fiducial markers for cryo-EM analysis of small proteins such as HCV E2 and identifies a new target epitope for vaccine development.

Hepatitis C virus (HCV) is a major medical health burden and the leading cause of chronic liver disease and cancer worldwide. More than 58 million people are chronically infected with HCV, with 1.5 million new infections occurring each year. An effective HCV vaccine is a major public health and medical need as recognized by the World Health Organization. However, due to the high variability of the virus and its ability to escape the immune response, HCV rapidly accumulates mutations, making vaccine development a formidable challenge. An effective vaccine must elicit broadly neutralizing antibodies (bnAbs) in a consistent fashion. After decades of studies from basic research through clinical development, the antigen of choice is considered the E1E2 envelope glycoprotein due to conserved, broadly neutralizing antigenic domains located in the constituent subunits of E1, E2, and the E1E2 heterodimeric complex itself. The challenge has been elicitation of robust humoral and cellular responses leading to broad virus neutralization due to the relatively low immunogenicity of this antigen. In view of this challenge, structure-based vaccine design approaches to stabilize key antigenic domains have been hampered due to the lack of E1E2 atomic-level resolution structures to guide them. Another challenge has been the development of a delivery platform in which a multivalent form of the antigen can be presented in order to elicit a more robust anti-HCV immune response. Recent nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both the cellular and humoral components of the immune system. This review focuses on recent advances in understanding the E1E2 heterodimeric structure to facilitate a rational design approach and the potential for development of a multivalent nanoparticle-based HCV E1E2 vaccine. Both aspects are considered important in the development of an effective HCV vaccine that can effectively address viral diversity and escape.

Over recent decades, therapeutic proteins have had widespread success in treating a myriad of diseases. Glycosylation, a near universal feature of this class of drugs, is a critical quality attribute that significantly influences the physical properties, safety profile and biological activity of therapeutic proteins. Optimizing protein glycosylation, therefore, offers an important avenue to developing more efficacious therapies. In this review, we discuss specific examples of how variations in glycan structure and glycoengineering impacts the stability, safety, and clinical efficacy of protein-based drugs that are already in the market as well as those that are still in preclinical development. We also highlight the impact of glycosylation on next generation biologics such as T cell-based cancer therapy and gene therapy.

‘Epivolve’ (epitope evolution) is an innovative paratope-evolving technology using a haptenated peptide or protein immunogen as a means of directing the in vivo immune response to specifically targeted sites at a one amino acid residue resolution. Guided by protein structural analysis, Epivolve technology was tested to develop site-directed neutralizing antibodies (nAbs) in a systematic fashion against the SARS-CoV-2 Receptor Binding Domain (RBD). Thirteen solvent-exposed sites covering the ACE2 receptor-binding interface were targeted. Immunogens composed of each targeted site were used to immunize rabbits in separate cohorts. In vivo site-directed immune responses against all 13 targets were demonstrated by B cell secreted IgG and recombinant IgG testing. One site, SL13 (Y505) which mutates from tyrosine to histidine in the SARS-CoV-2 Omicron variant, was chosen as a proof-of-concept (PoC) model for further functional monoclonal antibody development. Epivolve technology demonstrated the capabilities of generating pan-variant antibodies and nAbs against the SARS-CoV-2 primary strain and the Omicron variant.

Globally, more than 58 million people are chronically infected with Hepatitis C virus (HCV) with 1.5 million new infections occurring each year. An effective vaccine for HCV is therefore a major unmet medical and public health need. Since HCV rapidly accumulates mutations, vaccines must elicit the production of broadly neutralising antibodies (bnAbs) in a reproducible fashion. Decades of research have generated a number of HCV vaccine candidates. Based on the available data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice, but robust induction of humoral and cellular responses leading to virus neutralisation has not yet been achieved. One issue that has arisen in developing an HCV vaccine (and many other vaccines as well) is the platform used for antigen delivery. The majority of viral vaccine trials have employed subunit vaccines. However, subunit vaccines often have limited immunogenicity, as seen for HCV, and thus multiple formats must be examined in order to elicit a robust anti-HCV immune response. Nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both arms of the immune system. This review discusses the potential for development of a nanoparticle-based HCV E1E2 vaccine, with an emphasis on the potential benefits of such an approach along with the major challenges facing the incorporation of E1E2 into nanoparticulate delivery systems and how those challenges can be addressed.

Hepatitis C virus (HCV) is a major global health burden as the leading causative agent of chronic liver disease and hepatocellular carcinoma. While the main antigenic target for HCV-neutralizing antibodies is the membrane-associated E1E2 surface glycoprotein, the development of effective vaccines has been hindered by complications in the biochemical preparation of soluble E1E2 ectodomains. Here, we present a cryo-EM structure of an engineered, secreted E1E2 ectodomain of genotype 1b in complex with neutralizing antibodies AR4A, HEPC74, and IGH520. Structural characterization of the E1 subunit and C-terminal regions of E2 reveal an overall architecture of E1E2 that concurs with that observed for non-engineered full-length E1E2. Analysis of the AR4A epitope within a region of E2 that bridges between the E2 core and E1 defines the structural basis for its broad neutralization. Our study presents the structure of an E1E2 complex liberated from membrane via a designed scaffold, one that maintains all essential structural features of native E1E2. The study advances the understanding of the E1E2 heterodimer structure, crucial for the rational design of secreted E1E2 antigens in vaccine development. HCV vaccine development has been challenged by difficulties in the biochemical preparation of E1E2 ectodomains. Here, the authors structurally characterize an engineered soluble E1E2 ectodomain complexed with broadly neutralizing antibodies, revealing it adopts a native fold amenable for vaccine design.

Ebolavirus (EBOV) infection in humans is a severe and often fatal disease, which demands effective interventional strategies for its prevention and treatment. The available vaccines, which are authorized under exceptional circumstances, use viral vector platforms and have serious disadvantages, such as difficulties in adapting to new virus variants, reliance on cold chain supply networks, and administration by hypodermic injection. Microneedle (MN) patches, which are made of an array of micron-scale, solid needles that painlessly penetrate into the upper layers of the skin and dissolve to deliver vaccines intradermally, simplify vaccination and can thereby increase vaccine access, especially in resource-constrained or emergency settings. The present study describes a novel MN technology, which combines EBOV glycoprotein (GP) antigen with a polyphosphazene-based immunoadjuvant and vaccine delivery system (poly[di(carboxylatophenoxy)phosphazene], PCPP). The protein-stabilizing effect of PCPP in the microfabrication process enabled preparation of a dissolvable EBOV GP MN patch vaccine with superior antigenicity compared to a non-polyphosphazene polymer-based analog. Intradermal immunization of mice with polyphosphazene-based MN patches induced strong, long-lasting antibody responses against EBOV GP, which was comparable to intramuscular injection. Moreover, mice vaccinated with the MN patches were completely protected against a lethal challenge using mouse-adapted EBOV and had no histologic lesions associated with ebolavirus disease.

Significance Hepatitis C virus chronically infects approximately 1% of the world’s population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.