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Background and Aims Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpp) for neutralization assays. Methods We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpp. Neutralization sensitivity of these HCVpp varied widely. HCVpp clustered into 15 distinct groups based on patterns of relative sensitivity to seven broadly neutralizing monoclonal antibodies (bNAbs). We used these data to select a final panel of 15 antigenically representative HCVpp. Results Both the 65 and 15 HCVpp panels span four tiers of neutralization sensitivity, and neutralizing breadth measurements for seven bNAbs were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpp were independent of genetic distances between E1E2 clones. Conclusions Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity, rather than panels selected solely for genetic diversity. We propose that this multi-tier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.

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.

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., Chen et al. describe a rational design of subtilisin mutants that degrade active RAS by cleaving a conserved sequence in switch 2. They further modified the active site to be dependent on a cofactor to generate high target specificity. Proteases engineered to cleave this region degraded RAS in vitro and in cells with a promise of adaptability for other target proteins too.

Two well-defined synthetic polyphosphazene immunoadjuvants, PCPP and PCEP, were studied for their ability to potentiate the immune response to the hepatitis C virus (HCV) E2 glycoprotein antigen in vivo. We report that PCEP induced significantly higher serum neutralization and HCV-specific IgG titers in mice compared to other adjuvants used in the study: PCPP, Alum, and Addavax. PCEP also shifted the response toward the desirable balanced Th1/Th2 immunity, as evaluated by the antibody isotype ratio (IgG2a/IgG1). The in vivo results were analyzed in the context of antigen-adjuvant molecular interactions in the system and in vitro immunostimulatory activity of formulations. Asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) analysis showed that both PCPP and PCEP spontaneously self-assemble with the E2 glycoprotein with the formation of multimeric water-soluble complexes, which demonstrates the role of polyphosphazene macromolecules as vaccine delivery vehicles. Intrinsic in vitro immunostimulatory activity of polyphosphazene adjuvants, which was assessed using a mouse macrophage cell line, revealed comparable activities of both polymers and did not provide an explanation of their in vivo performance. However, PCEP complexes with E2 displayed greater stability against agglomeration and improved in vitro immunostimulatory activity compared to those of PCPP, which is in line with superior in vivo performance of PCEP. The results emphasize the importance of often neglected antigen-polyphosphazene self-assembly mechanisms in formulations, which can provide important insights on their in vivo behavior and facilitate the establishment of a structure-activity relationship for this important class of immunoadjuvants.

Resiquimod or R848 (RSQD) is a Toll-like receptor (TLR) 7/8 agonist which shows promise as vaccine adjuvant due to its potential to promote highly desirable cellular immunity. The development of this small molecule in the field to date has been largely impeded by its rapid in vivo clearance and lack of association with vaccine antigens. Here, we report a multimeric TLR 7/8 construct of nano-scale size, which results from a spontaneous self-assembly of RSQD with a water-soluble clinical-stage polymer - poly[di(carboxylatophenoxy)phosphazene] (PCPP). The formation of ionically paired construct (PCPP-R) and a ternary complex, which also includes Hepatitis C virus (HCV) antigen, has been demonstrated by dynamic lights scattering (DLS), turbidimetry, fluorescence spectroscopy, asymmetric flow field flow fractionation (AF4), and 1H NMR spectroscopy methods. The resulting supramolecular assembly PCPP-R enabled superior immunostimulation in cellular assays (mouse macrophage reporter cell line) and displayed improved in vitro hemocompatibility (human erythrocytes). In vivo studies demonstrated that PCPP-R adjuvanted HCV formulation induced higher serum neutralization titers in BALB/c mice and shifted the response towards desirable cellular immunity, as evaluated by antibody isotype ratio (IgG2a/IgG1) and ex vivo analysis of cytokine secreting splenocytes (higher levels of interferon gamma (IFN-γ) single and tumor necrosis factor alpha (TNF-α)/IFN-γ double producing cells). The non-covalent multimerization approach stands in contrast to previously suggested RSQD delivery methods, which involve covalent conjugation or encapsulation, and offers a flexible methodology that can be potentially integrated with other parenterally administered drugs.

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.

An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.

Resiquimod or R848 (RSQD) is a Toll-like receptor (TLR) 7/8 agonist which shows promise as vaccine adjuvant due to its potential to promote highly desirable cellular immunity. The development of this small molecule in the field to date has been largely impeded by its rapid in vivo clearance and lack of association with vaccine antigens. Here, we report a multimeric TLR 7/8 construct of nano-scale size, which results from a spontaneous self-assembly of RSQD with a water-soluble clinical-stage polymer - poly[di(carboxylatophenoxy)phosphazene] (PCPP). The formation of ionically paired construct (PCPP-R) and a ternary complex, which also includes Hepatitis C virus (HCV) antigen, has been demonstrated by dynamic lights scattering (DLS), turbidimetry, fluorescence spectroscopy, asymmetric flow field flow fractionation (AF4), and (1)H NMR spectroscopy methods. The resulting supramolecular assembly PCPP-R enabled superior immunostimulation in cellular assays (mouse macrophage reporter cell line) and displayed improved in vitro hemocompatibility (human erythrocytes). In vivo studies demonstrated that PCPP-R adjuvanted HCV formulation induced higher serum neutralization titers in BALB/c mice and shifted the response towards desirable cellular immunity, as evaluated by antibody isotype ratio (IgG2a/IgG1) and ex vivo analysis of cytokine secreting splenocytes (higher levels of interferon gamma (IFN-γ) single and tumor necrosis factor alpha (TNF-α)/IFN-γ double producing cells). The non-covalent multimerization approach stands in contrast to previously suggested RSQD delivery methods, which involve covalent conjugation or encapsulation, and offers a flexible methodology that can be potentially integrated with other parenterally administered drugs.

Achieving intracellular delivery of protein therapeutics within cells remains a significant challenge. Although custom formulations are available for some protein therapeutics, the development of non-toxic delivery systems that can incorporate a variety of active protein cargo and maintain their stability, is a topic of great relevance. This study utilized ionic polyphosphazenes (PZ) that can assemble into supramolecular complexes through non-covalent interactions with different types of protein cargo. We tested a PEGylated graft copolymer (PZ-PEG) and a pyrrolidone containing linear derivative (PZ-PYR) for their ability to intracellularly deliver FITC-avidin, a model protein. In endothelial cells, PZ-PYR/protein exhibited both faster internalization and higher uptake levels than PZ-PEG/protein, while in cancer cells both polymers achieved similar uptake levels over time, although the internalization rate was slower for PZ-PYR/protein. Uptake was mediated by endocytosis through multiple mechanisms, PZ-PEG/avidin colocalized more profusely with endo-lysosomes, and PZ-PYR/avidin achieved greater cytosolic delivery. Consequently, a PZ-PYR-delivered anti-F-actin antibody was able to bind to cytosolic actin filaments without needing cell permeabilization. Similarly, a cell-impermeable Bax-BH3 peptide known to induce apoptosis, decreased cell viability when complexed with PZ-PYR, demonstrating endo-lysosomal escape. These biodegradable PZs were non-toxic to cells and represent a promising platform for drug delivery of protein therapeutics.

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

Copyright © 2020 American Society for Microbiology. 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 broadly neutralizing antibodies (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.IMPORTANCE Hepatitis C virus infects approximately 1% of the world's population, and no vaccine is currently available. Due to the high variability of HCV and its ability to actively escape the immune response, a goal of HCV vaccine design is to induce neutralizing antibodies that target conserved epitopes. Here, we performed structure-based design of several epitopes of the HCV E2 envelope glycoprotein to engineer its antigenic properties. Designs were tested in vitro and in vivo, demonstrating alteration of the E2 antigenic profile in several cases, and one design led to improvement of cross-neutralization of heterologous viruses. This represents a proof of concept that rational engineering of HCV envelope glycoproteins can be used to modulate E2 antigenicity and optimize a vaccine for this challenging viral target.

The severity of the 2014–2016 ebolavirus outbreak in West Africa expedited clinical development of therapeutics and vaccines though the countermeasures on hand were largely monospecific and lacked efficacy against other ebolavirus species that previously emerged. Recent studies indicate that ebolavirus glycoprotein (GP) fusion loops are targets for cross-protective antibodies. Here we report the 3.72 Å resolution crystal structure of one such cross-protective antibody, CA45, bound to the ectodomain of Ebola virus (EBOV) GP. The CA45 epitope spans multiple faces of the fusion loop stem, across both GP1 and GP2 subunits, with ~68% of residues identical across > 99.5% of known ebolavirus isolates. Extensive antibody interactions within a pan-ebolavirus small-molecule inhibitor binding cavity on GP define this cavity as a novel site of immune vulnerability. The structure elucidates broad ebolavirus neutralization through a highly conserved epitope on GP and further enables rational design and development of broadly protective vaccines and therapeutics., The Ebola virus glycoprotein is a target for cross-protective antibodies. Here, Janus et al. report the crystal structure of the antigen-binding fragment of a pan-reactive antibody bound to a conserved epitope of the glycoprotein, facilitating rational design of cross-protective vaccines and therapeutics.

Nanoparticulate and water-soluble formulations of ionic polyphosphazenes and protein cargo - lysozyme (LYZ) were prepared by their self-assembly in aqueous solutions at near physiological pH (pH 7.4) in the presence and absence of an ionic cross-linker – spermine tetrahydrochloride. Efficiency of LYZ encapsulation, physico-chemical characteristics of formulations, and the effect of reaction parameters were investigated using asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) methods. The effect of both polymer formulations on encapsulated LYZ was evaluated using soluble oligosaccharide substrate, whereas their ability to present the protein to cellular surfaces was assessed by measuring enzymatic activity of encapsulated LYZ against Micrococcus lysodeikticus cells. It was found that both soluble and cross-linked polymer matrices reduce lysis of bacterial cells by LYZ, whereas activity of encapsulated protein against oligosaccharide substrate remained practically unchanged indicating no adverse effect of polyphosphazene on protein integrity. Moreover, nanoparticulate formulations display distinctly different behavior in cellular assays when compared to their soluble counterparts. LYZ encapsulated in polyphosphazene nanoparticles shows approximately 2.5-fold higher activity in its ability to lyse cells as compared with water-soluble LYZ-PCPP formulations. A new approach to PEGylation of polyphosphazene nanoparticles was also developed. The method utilizes a new ionic polyphosphazene derivative, which contains graft (polyethylene glycol) chains. PEGylation allows for an improved control over the size of nanoparticles and broader modulation of their cross-linking density, while still permitting for protein presentation to cellular substrates.

The development of a vaccine for hepatitis C virus (HCV) remains a global health challenge. A major challenge for vaccine development is focusing the immune response on conserved regions of the HCV envelope protein, E2, capable of eliciting neutralizing antibodies. Modification of E2 by glycosylation might influence the immunogenicity of E2. Accordingly, we performed molecular and immunogenic comparisons of E2 produced in mammalian and insect cells. Mass spectrometry demonstrated that the predicted glycosylation sites were utilized in both mammalian and insect cell E2, although the glycan types in insect cell E2 were smaller and less complex. Mouse immunogenicity studies revealed similar polyclonal antibody responses. However, insect cell E2 induced stronger neutralizing antibody responses against the homologous isolate used in the vaccine, albeit the two proteins elicited comparable neutralization titers against heterologous isolates. A more productive approach for vaccine development may be complete deletion of specific glycans in the E2 protein., The development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic, and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex- and high-mannose-type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose-type glycans. Mass spectrometry demonstrated that all 11 predicted N-glycosylation sites were utilized in both HEK293- and Sf9-derived sE2, but that N-glycans in insect sE2 were on average smaller and less complex. Both proteins bound CD81 and were recognized by conformation-dependent antibodies. Mouse immunogenicity studies revealed that similar polyclonal antibody responses were generated against antigenic domains A to E of E2. Although neutralizing antibody titers showed that Sf9-derived sE2 induced moderately stronger responses than did HEK293-derived sE2 against the homologous HCV H77c isolate, the two proteins elicited comparable neutralization titers against heterologous isolates. Given that global alteration of HCV E2 glycosylation by expression in different hosts did not appreciably affect antigenicity or overall immunogenicity, a more productive approach to increasing the antibody response to neutralizing epitopes may be complete deletion, rather than just modification, of specific N-glycans proximal to these epitopes. IMPORTANCE The development of a vaccine for hepatitis C virus (HCV) remains a global health challenge. A major challenge for vaccine development is focusing the immune response on conserved regions of the HCV envelope protein, E2, capable of eliciting neutralizing antibodies. Modification of E2 by glycosylation might influence the immunogenicity of E2. Accordingly, we performed molecular and immunogenic comparisons of E2 produced in mammalian and insect cells. Mass spectrometry demonstrated that the predicted glycosylation sites were utilized in both mammalian and insect cell E2, although the glycan types in insect cell E2 were smaller and less complex. Mouse immunogenicity studies revealed similar polyclonal antibody responses. However, insect cell E2 induced stronger neutralizing antibody responses against the homologous isolate used in the vaccine, albeit the two proteins elicited comparable neutralization titers against heterologous isolates. A more productive approach for vaccine development may be complete deletion of specific glycans in the E2 protein.

Despite the development of direct-acting antivirals (DAAs), hepatitis C virus (HCV) infection remains a major cause for liver disease and cancer worldwide. Entry inhibitors block virus host cell entry and, therefore, prevent establishment of chronic infection and liver disease. Due to their unique mechanism of action, entry inhibitors provide an attractive antiviral strategy in organ transplantation. In this study, we developed an innovative approach in preventing HCV infection using a synergistic combination of a broadly neutralizing human monoclonal antibody (HMAb) targeting the HCV E2 protein and a host-targeting anti-claudin 1 (CLDN1) humanized monoclonal antibody. An in vivo proof-of-concept study in human liver-chimeric FRG-NOD mice proved the efficacy of the combination therapy at preventing infection by an HCV genotype 1b infectious serum. While administration of individual antibodies at lower doses only showed a delay in HCV infection, the combination therapy was highly protective. Furthermore, the combination proved to be effective in preventing infection of primary human hepatocytes by neutralization-resistant HCV escape variants selected during liver transplantation, suggesting that a combination therapy is suited for the neutralization of difficult-to-treat variants. In conclusion, our findings suggest that the combination of two HMAbs targeting different steps of virus entry improves treatment efficacy while simultaneously reducing treatment duration and costs. Our approach not only provides a clinical perspective to employ HMAb combination therapies to prevent graft re-infection and its associated liver disease but may also help to alleviate the urgent demand for organ transplants by allowing the transplantation of organs from HCV-positive donors.

Novel oppositely charged polyphosphazene polyelectrolytes containing grafted poly(ethylene glycol) (PEG) chains were synthesized as modular components for the assembly of biodegradable PEGylated protein delivery vehicles. These macromolecular counterparts, which contained either carboxylic acid or tertiary amino groups, were then formulated at near physiological conditions into supramolecular assemblies of nanoscale level, below 100 nm. Nanocomplexes with electroneutral surface charge, as assessed by zeta potential measurements, were stable in aqueous solutions, which suggests their compact polyelectrolyte complex "core"-hydrophilic PEG "shell" structure. Investigation of PEGylated polyphosphazene nanocomplexes as agents for noncovalent PEGylation of the therapeutic protein l-asparaginase (L-ASP) in vitro demonstrated their ability to dramatically reduce protein antigenicity, as measured by antibody binding using enzyme linked immunosorbent assay (ELISA). Encapsulation in nanocomplexes did not affect enzymatic activity of L-ASP, but improved its thermal stability and proteolytic resistance. Gel permeation chromatography (GPC) experiments revealed that all synthesized polyphosphazenes exhibited composition controlled hydrolytic degradability in aqueous solutions at neutral pH and showed greater stability at lower temperatures. Overall, novel hydrolytically degradable polyphosphazene polyelectrolytes capable of spontaneous self-assembly into PEGylated nanoparticulates in aqueous solutions can potentially enable a simple and effective approach to modifying therapeutic proteins without the need for their covalent modification.

The ability to use structure-based design and engineering to control the molecular shape and reactivity of an immunogen to induce protective responses shows great promise, along with corresponding advancements in vaccine testing and evaluation systems. We describe in this review new paradigms for the development of a B cell-based HCV vaccine. Advances in test systems to measure in vitro and in vivo antibody-mediated virus neutralization include retroviral pseudotype particles expressing HCV E1E2 glycoproteins (HCVpp), infectious cell culture-derived HCV virions (HCVcc), and surrogate animal models mimicking acute HCV infection. Their applications have established the role of broadly neutralizing antibodies to control HCV infection. However, the virus has immunogenic regions in the viral envelope glycoproteins that are associated with viral escape or non-neutralizing antibodies. These regions serve as immunologic decoys that divert the antibody response from less prominent conserved regions mediating virus neutralization. This review outlines the immunogenic regions on E2, which are roughly segregated into the hypervariable region 1 (HVR1), and five clusters of overlapping epitopes designated as antigenic domains A-E. Understanding the molecular architecture of conserved neutralizing epitopes within these antigenic domains, and how other antigenic regions or decoys deflect the immune response from these conserved regions will provide a roadmap for the rational design of an HCV vaccine.

A series of biodegradable drug delivery polymers with intrinsic multifunctionality have been designed and synthesized utilizing polyphosphazene macromolecular engineering approach. Novel water-soluble polymers, which contain carboxylic acid and pyrrolidone moieties attached to inorganic phosphorus-nitrogen backbone, were characterized by a suite of physico-chemical methods to confirm their structure, composition, and molecular sizes. All synthesized polyphosphazenes displayed composition dependent hydrolytic degradability in aqueous solutions at neutral pH. Their formulations were stable at lower temperatures, potentially indicating adequate shelf life, but were characterized by accelerated degradation kinetics at elevated temperatures, including 37°C. It was found that synthesized polyphosphazenes are capable of environmentally triggered self-assembly to produce nanoparticles with narrow polydispersity in the size range between 150 and 700 nm. Protein loading capacity of copolymers has been validated via their ability to non-covalently bind avidin without altering its biological functionality. Acid induced membrane disruptive activity of polyphosphazenes has been established with an onset corresponding to endosomal pH range and being dependent on polymer composition. The synthesized polyphosphazenes facilitated cell-surface interaction followed by time-dependent, vesicular mediated, and saturable internalization of a model protein cargo into cancer cells, demonstrating potential for intracellular delivery.

Aluminum salts are the most widely used vaccine adjuvants, and phosphate is known to modulate antigen-adjuvant interactions. Here we report an unexpected role for phosphate buffer in an anthrax vaccine (SparVax) containing recombinant protective antigen (rPA) and aluminum oxyhydroxide (AlOH) adjuvant (Alhydrogel). Phosphate ions bind to AlOH to produce an aluminum phosphate surface with a reduced rPA adsorption coefficient and binding capacity. However, these effects continued to increase as the free phosphate concentration increased, and the binding of rPA changed from endothermic to exothermic. Crucially, phosphate restored the thermostability of bound rPA so that it resembled the soluble form, even though it remained tightly bound to the surface. Batches of vaccine with either 0.25 mM (subsaturated) or 4 mM (saturated) phosphate were tested in a disease model at batch release, which showed that the latter was significantly more potent. Both formulations retained their potency for 3 years. The strongest aluminum adjuvant effects are thus likely to be via weakly attached or easily released native-state antigen proteins.

Direct acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to employ neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified a HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more importantly, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity matured HMAb mediates virus neutralization in part by inducing conformational change to the targeted epitope and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation. Conclusions: The findings suggest that a single broadly neutralizing antibody can prevent acute HCV infection without inducing RAVs and may complement DAAs to reduce the emergence of RAVs. This article is protected by copyright. All rights reserved.

The E2 envelope glycoprotein is the primary target of human neutralizing antibody response against hepatitis C virus (HCV), and is thus a major focus of vaccine and immunotherapeutics efforts. There is emerging evidence that E2 is a highly complex, dynamic protein with residues across the protein that are modulating antibody recognition, local and global E2 stability, and viral escape. To comprehensively map these determinants, we performed global E2 alanine scanning with a panel of 16 human monoclonal antibodies (hmAbs), resulting in an unprecedented dataset of the effects of individual alanine substitutions across the E2 protein (355 positions) on antibody recognition. Analysis of shared energetic effects across the antibody panel identified networks of E2 residues involved in antibody recognition and local and global E2 stability, as well as predicted contacts between residues across the entire E2 protein. Further analysis of antibody binding hotspot residues defined groups of residues essential for E2 conformation and recognition for all 14 conformationally dependent E2 antibodies and subsets thereof, as well as residues that enhance antibody recognition when mutated to alanine, providing a potential route to engineer E2 vaccine immunogens. By incorporating E2 sequence variability, we found a number of E2 polymorphic sites that are responsible for loss of neutralizing antibody binding. These data and analyses provide fundamental insights into antibody recognition of E2, highlighting the dynamic and complex nature of this viral envelope glycoprotein, and can serve as a reference for development and rational design of E2-targeting vaccines and immunotherapeutics.

Two macromolecular immunoadjuvants, poly[di(carboxylatophenoxy)phosphazene], PCPP, and poly[di(carboxylatoethylphenoxy)phosphazene], PCEP, have been investigated for their molecular interactions with model and biopharmaceutically important proteins in solutions, as well as for their TLR stimulatory effects and pH-dependent membrane disruptive activity in cellular assays. Solution interactions between polyphosphazenes and proteins, including antigens and soluble immune receptor proteins, have been studied using Asymmetric Flow Field Flow Fractionation (AF4) and Dynamic Light Scattering (DLS) at near physiological conditions: phosphate buffered saline, pH 7.4. Polyphosphazenes demonstrated selectivity in their molecular interactions with various proteins, but displayed strong binding with all vaccine antigens tested in the present study. It was found that both PCPP and PCEP showed strong avidity to soluble immune receptor proteins, such as Mannose Receptor (MR) and certain Toll-Like Receptor (TLR) proteins. Studies on TLR stimulation in vitro using HEK293 cells with overexpressed human TLRs revealed activation of TLR7, TLR8, and TLR9 signaling pathways, albeit with some nonspecific stimulation, for PCPP and the same pathways plus TLR3 for PCEP. Finally, PCEP, but not PCPP, demonstrated pH-dependent membrane disruptive activity in the pH range corresponding to the pH environment of early endosomes, which may play a role in a cross-presentation of antigenic proteins.

Humoral immunity is the primary correlate of protection for most preventive vaccines. For hepatitis C virus (HCV), early animal studies provided support for the importance of virus neutralizing antibodies to facilitate clearance of infection. The development of retroviral pseudotype particles expressing HCV E1E2 glycoproteins (HCVpp), infectious cell culture-derived HCV virions (HCVcc), and small animal models of acute HCV infection have made possible the measurement of in vitro and in vivo antibody-mediated virus neutralization. Their applications in multiple studies clearly established the importance of neutralizing antibodies in controlling HCV infection. However, the virus has devised a number of decoys of highly immunogenic regions associated with viral escape or non-neutralizing antibodies that deflect the immune response from less immunogenic conserved regions mediating virus neutralization that are not associated with viral escape or escape with compromised fitness. The focus of this review is on the immunogenic determinants on E2, which are roughly segregated into the hypervariable region 1 (HVR1), and five clusters of overlapping epitopes, designated as antigenic domains A-E. A detailed understanding of conserved neutralizing epitopes within these antigenic domains that are not associated with escape and how other antigenic regions or decoys serve as diversions of the immune response and/or elicit antibodies that negatively modulate broadly neutralizing antibodies will provide a roadmap in rational design for an HCV vaccine.

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. A challenge for HCV vaccine development is to identify conserved epitopes able to elicit protective antibodies against this highly diverse virus. Glycan shielding is a mechanism by which HCV masks such epitopes on its E2 envelope glycoprotein. Antibodies to the E2 region comprising residues 412-423 (E2(412-423)) have broadly neutralizing activities. However, an adaptive mutation in this linear epitope, N417S, is associated with a glycosylation shift from Asn-417 to Asn-415 that enables HCV to escape neutralization by mAbs such as HCV1 and AP33. By contrast, the human mAb HC33.1 can neutralize virus bearing the N417S mutation. To understand how HC33.1 penetrates the glycan shield created by the glycosylation shift to Asn-415, we determined the structure of this broadly neutralizing mAb in complex with its E2(412-423) epitope to 2.0 Å resolution. The conformation of E2(412-423) bound to HC33.1 is distinct from the β-hairpin conformation of this peptide bound to HCV1 or AP33, because of disruption of the β-hairpin through interactions with the unusually long complementarity-determining region 3 of the HC33.1 heavy chain. Whereas Asn-415 is buried by HCV1 and AP33, it is solvent-exposed in the HC33.1-E2(412-423) complex, such that glycosylation of Asn-415 would not prevent antibody binding. Furthermore, our results highlight the structural flexibility of the E2(412-423) epitope, which may serve as an immune evasion strategy to impede induction of antibodies targeting this site by reducing its antigenicity.

We developed a stringently regulated expression system for mammalian cells that uses (i) the RNA polymerase, phi 10 promoter, and T phi transcriptional terminator of bacteriophage T7; (ii) the lac repressor, lac operator, rho-independent transcriptional terminators and the gpt gene of Escherichia coli; (iii) the RNA translational enhancer of encephalomyocarditis virus; and (iv) the genetic background of vaccinia virus. In cells infected with the recombinant vaccinia virus, reporter beta-galactosidase synthesis was not detected in the absence of inducer. An induction of at least 10,000- to 20,000-fold occurred upon addition of isopropyl beta-D-thiogalactopyranoside or by temperature elevation from 30 to 37 degrees C using a temperature-sensitive lac repressor. Regulated synthesis of the secreted and highly glycosylated human immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were approximately 2 mg per 10(8) cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.

Inhalation anthrax is a potentially lethal form of disease resulting from exposure to aerosolizedBacillus anthracisspores. Over the last decade, incidents spanning from the deliberate mailing ofB. anthracisspores to incidental exposures in users of illegal drugs have highlighted the importance of developing new medical countermeasures to protect people who have been exposed to “anthrax spores” and are at risk of developing disease. The New Zealand White rabbit (NZWR) is a well-characterized model that has a pathogenesis and clinical presentation similar to those seen in humans. This article reports how the NZWR model was adapted to evaluate postexposure prophylaxis using a recombinant protective antigen (rPA) vaccine in combination with an oral antibiotic, levofloxacin. NZWRs were exposed to multiples of the 50% lethal dose (LD50) ofB. anthracisspores and then vaccinated immediately (day 0) and again on day 7 postexposure. Levofloxacin was administered daily beginning at 6 to 12 h postexposure for 7 treatments. Rabbits were evaluated for clinical signs of disease, fever, bacteremia, immune response, and survival. A robust immune response (IgG anti-rPA and toxin-neutralizing antibodies) was observed in all vaccinated groups on days 10 to 12. Levofloxacin plus either 30 or 100 μg rPA vaccine resulted in a 100% survival rate (18 of 18 per group), and a vaccine dose as low as 10 μg rPA resulted in an 89% survival rate (16 of 18) when used in combination with levofloxacin. In NZWRs that received antibiotic alone, the survival rate was 56% (10 of 18). There was no adverse effect on the development of a specific IgG response to rPA in unchallenged NZWRs that received the combination treatment of vaccine plus antibiotic. This study demonstrated that an accelerated two-dose regimen of rPA vaccine coadministered on days 0 and 7 with 7 days of levofloxacin therapy results in a significantly greater survival rate than with antibiotic treatment alone. Combination of vaccine administration and antibiotic treatment may be an effective strategy for treating a population exposed to aerosolizedB. anthracisspores.

It has been proposed that proteases are important in endothelial cell behavior. We examined the contribution of the gelatinase/type IV collagenase system in an in vitro model of endothelial differentiation. Human umbilical vein endothelial cells rapidly align and form networks of tubes when cultured on a basement membrane preparation, Matrigel. Zymograms of culture supernates demonstrate a 72-kD and a 92-kD gelatinase activity; the cells produce most of the 72-kD gelatinase, whereas the 92-kD activity is derived entirely from the Matrigel. Addition of antibodies against type IV gelatinase/collagenase decreases the area of the tube network. Both tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, similarly decrease tube formation when added to cultures. Conversely, exogenous recombinant 72-kD gelatinase increases tube-forming activity. The effects of the anti-gelatinase antibodies and the TIMPs are not additive. Inhibition by either antibodies or TIMPs is greatest when they are added at culture initiation, suggesting that the protease activity is important in the early steps of morphogenesis. However, culture of the cells on Matrigel does not increase early expression of mRNA for the 72-kD gelatinase. Expression of message for the enzyme actually decreases during the course of the assay, while transcription of mRNAs for TIMPs increases, further supporting the concept that collagenases facilitate an early event in tube formation. These data demonstrate that gelatinase/type IV collagenase activity is important in endothelial cell morphogenesis on Matrigel, and suggest a role for collagenases in formation of new capillaries in vivo.

The 72-kDa gelatinase/type IV collagenase, a metalloproteinase thought to play a role in metastasis and in angiogenesis, forms a noncovalent stoichiometric complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2), a potent inhibitor of enzyme activity. To define the regions of the 72-kDa gelatinase responsible for TIMP-2 binding, a series of NH2- and COOH-terminal deletions of the enzyme were constructed using the polymerase chain reaction technique. The full-length and the truncated enzymes were expressed in a recombinant vaccinia virus mammalian cell expression system (Vac/T7). Two truncated enzymes ending at residues 425 (delta 426-631) and 454 (delta 455-631) were purified. Like the full-length recombinant 72-kDa gelatinase, both COOH-terminally truncated enzymes were activated with organomercurial and digested gelatin and native collagen type IV. In contrast to the full-length enzyme, delta 426-631 and delta 455-631 enzymes were less sensitive to TIMP-2 inhibition requiring 10 mol of TIMP-2/mol of enzyme to achieve maximal inhibition of enzymatic activity. The activated but not the latent forms of the delta 426-631 and delta 455-631 proteins formed a complex with TIMP-2 only when excess molar concentrations of inhibitor were used. We also expressed the 205-amino acid COOH-terminal fragment, delta 1-426, and found that it binds TIMP-2. In addition, a truncated version of the 72-kDa gelatinase lacking the NH2-terminal 78 amino acids (delta 1-78) of the proenzyme retained the ability to bind TIMP-2. These studies demonstrate that 72-kDa gelatinases lacking the COOH-terminal domain retain full enzymatic activity but acquire a reduced sensitivity to TIMP-2 inhibition. These data suggest that both the active site and the COOH-terminal tail of the 72-kDa gelatinase independently and cooperatively participate in TIMP-2 binding.

Three IgG1 human monoclonal antibodies (MAbs) directed against conformational epitopes of the gp120 envelope protein of HIV-1 were produced, as was a single human MAb to a linear epitope spanning amino acids 487-509 in the C-terminal portion of gp120. All three conformation-dependent MAbs reacted optimally with recombinant gp120 (rgp120) captured on plastic via its carbohydrate moieties with Concanavalin A. These MAbs were able to block the interaction between recombinant CD4 (rCD4) and rgp120; they were also able to achieve 50% neutralization of HTLV-IIIB and MN strains of HIV-1 in a concentration range of 0.5-12.8 micrograms/mL. The MAb to the linear determinant is the first reported human MAb specific for the immunodominant portion of gp120; this MAb was most reactive with rgp120 when it was coated directly on plastic. It could neither inhibit rCD4-rgp120 binding nor neutralize either HTLV-IIIB or MN. The binding affinities of the four human MAbs for rgp120 in solution, reflected by their dissociation constants (Kd), ranged from 0.5 x 10(-8) to 7.5 x 10(-8) M.

BCG, a live attenuated tubercle bacillus, is the most widely used vaccine in the world and is also a useful vaccine vehicle for delivering protective antigens of multiple pathogens. Extrachromosomal and integrative expression vectors carrying the regulatory sequences for major BCG heat-shock proteins have been developed to allow expression of foreign antigens in BCG. These recombinant BCG strains can elicit long-lasting humoral and cellular immune responses to foreign antigens in mice.

The role that individual determinants play in modulating the immune response of an organism to a pathogen is often obscured because of the complexity of the pathogen. In order to gain a better appreciation of the role of individual determinants in the immune response, a pathogen may be dissociated into smaller components, for example peptides representing specific epitopes. These isolated components are often poorly immunogenic and historically have required the use of adjuvants to stimulate antibody production. This report defines the minimal essential requirements for antibody production to a peptide in this system. These are the ability to stimulate both B- and T-helper lymphocytes, anchorage in a phospholipid complex and multivalency within the complex. When these conditions are met, no additional adjuvants are necessary. This procedure has allowed us to identify three distinct T-helper cell epitopes from HIV gp160. In addition, this information has been used to produce a simple, totally synthetic and highly immunogenic preparation for the production of antibodies to peptides.

The hepatitis E virus (HEV) open reading frame-2 (ORF-2) is predicted to encode a 71-kDa putative capsid protein involved in virus particle formation. When insect Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus containing the entire ORF-2 sequence, two types of recombinant proteins were produced; an insoluble protein of 73 kDa and a soluble protein of 62 kDa. The 62-kDa species was shown to be a proteolytic cleavage product of the 73-kDa protein. N-terminal sequence analysis of the 62-kDa protein indicated that it lacked the first 111 amino acids that are present in the full-length 73-kDa protein. A soluble 62-kDa protein was produced without the proteolytic processing by inserting the coding sequence of amino acids 112 to 660 of ORF-2 in a baculovirus expression vector and using the corresponding virus to infect Sf9 cells. The two recombinant 62-kDa proteins made by different mechanisms displayed immunoreactivities very compatible to each other. The 62-kDa proteins obtained by both proteolytic processing and reengineering demonstrated much higher sensitivities in detecting anti-HEV antibodies in human sera than the antigens made from bacteria, as measured by enzyme-linked immunosorbent assay. The data suggest that the soluble 62-kDa protein made from insect cells contains additional epitopes not present in recombinant proteins made from bacteria. Therefore, the 62-kDa protein may be useful for HEV diagnostic improvement and vaccine development. The reengineered construct allows for the consistent large-scale production of the soluble 62-kDa protein without proteolytic processing.

The dynamics of plasma viremia were explored in a group of 12 simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) that had received prior immunization with either nonrecombinant or trivalent (gag-pol, env) SIV-recombinant vaccinia viruses. Three distinct patterns of viral replication observed during and following primary viremia accounted for significant differences in survival times. High-level primary plasma viremia with subsequently increasing viremia was associated with rapid progression to AIDS (n = 2). A high-level primary plasma virus load with a transient decline and subsequent progressive increase in viremia in the post-acute phase of infection was associated with progression to AIDS within a year (n = 6). Low levels of primary plasma viremia followed by sustained restriction of virus replication were associated with maintenance of normal lymphocyte subsets and intact lymphoid architecture (n = 4), reminiscent of the profile observed in human immunodeficiency virus type 1-infected long-term nonprogressors. Three of four macaques that showed this pattern had been immunized with an SIV recombinant derived from the attenuated vaccinia virus, modified vaccinia virus Ankara. These data link the dynamics and extent of virus replication to disease course and suggest that sustained suppression of virus promotes long-term, asymptomatic survival of SIV-infected macaques. These findings also suggest that vaccine modulation of host immunity may have profound beneficial effects on the subsequent disease course, even if sterilizing immunity is not achieved.

The use of a recombinant virus containing a heterologous gene of another microorganism as a live vaccine was suggested more than 10 years ago (Mackett et al, 1982; Panicali and Paoletti, 1982). Vaccinia virus was considered for such a purpose because of its success as a smallpox vaccine and ease and economy of production, distribution and administration (Fermer et al., 1988). The extensive experimental use of recombinant vaccinia viruses was facilitated by the construction of plasmid transfer vectors containing a vaccinia virus promoter, one or more convenient restriction endonuclease sites for inserting a foreign gene, flanking DNA sequences for homologous recombination into a non-essential site of the vaccinia virus genome and for selection and/or screening of recombinant viruses (Chakrabarti et al., 1985; Mackett et al., 1984). Humoral and cell mediated immune responses to an expressed foreign protein and protection of experimental animals against challenge with the corresponding pathogen were demonstated in a variety of animal model systems (Cox et al., 1992; Moss, 1991).