Harnessing Liquid Crystal Sensors for High-Throughput Real-Time Detection of Structural Changes in Lysozyme during Refolding Processes.

Despite the rapid advances in process analytical technology, the assessment of protein refolding efficiency has largely relied on off-line protein-specific assays and/or chromatographic procedures such as reversed-phase high-performance liquid chromatography and size exclusion chromatography. Due to the inherent time gap pertaining to traditional methods, exploring optimum refolding conditions for many recombinant proteins, often expressed as insoluble inclusion bodies, has proven challenging. The present study describes a novel protein refolding sensor that utilizes liquid crystals (LCs) to discriminate varying protein structures during unfolding and refolding. An LC layer containing 4-cyano-4'-pentylbiphenyl (5CB) intercalated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is used as a sensing platform, and its proof-of-concept performance is demonstrated using lysozyme as a model protein. As proteins unfold or refold, a local charge fluctuation at their surfaces modulates their interaction with zwitterionic phospholipid DOPE. This alters the alignment of DOPE molecules at the aqueous/LC interface, affecting the orientational ordering of bulk LC (i.e., homeotropic to planar for refolding and planar to homeotropic for unfolding). Differential polarized optical microscope images of the LC layer are subsequently generated, whose brightness directly linked to conformational changes of lysozyme molecules is quantified by gray scale analysis. Importantly, our LC-based refolding sensor is compatible with diverse refolding milieus for real-time analysis of lysozyme refolding and thus likely to facilitate the refolding studies of many proteins, especially those lacking a method to determine structure-dependent biological activity.

Engineered Design of the E-Helix Structure on Ferritin Nanoparticles.

Insertion of an immunogenic epitope at the C-terminus of ferritin has shown the potential to produce a stable and efficacious vaccine. There is however limited understanding of how C-terminus insertion affects ferritin protein stability. The E-helix at the C-terminus has attracted interest because there are contradictory reports as to whether it has a role in protein stabilization. Here, we report, for the first time, combining molecular dynamics simulation (MDS) with experiment to engineer the design of the E-helix at the C-terminus of engineered human ferritin heavy chain (F1) inserted with Epstein-Barr nuclear antigen 1 (EBNA1, E1) and flexible linker (L3) residues (to afford F1L3E1). Hot spots on the E-helix of the C-terminus were predicted by MDS at aa 167 (Glu) and aa 171 (Asp). Five (5) variants of F1L3E1 were constructed by considering hot spots and alteration of electrostatic or hydrophobic interfaces, namely, (1) C1, hot spots substituted with noncharged residue Gln; (2) C2, hot spots substituted with positively charged residue Arg; (3) C3, hydrophobic residues substituted with the most hydrophobic residues Val and Ile; (4) C4, hydrophobic residues substituted with the most hydrophilic residues Gln and Asn; and (5) C5, a heptad repeat structure in the E-helix disrupted by substituting "a" and "d" heptad residues with noncharged polar residue Gln. It was found that the E-helix is essential to maintain integrated protein stability and that changing the hydrophobic interface (C3 and C4) had more significant effects on protein folding and stability than changing the electrostatic interface (C1 and C2). It was confirmed by both MDS and experiment that variants C1, C2, and C5 were able to fold to form stable conformational structures with protein surface hydrophobicity similar to that of F1L3E1. However, they are less thermally stable than F1L3E1. Significant changes in hydrophobicity drove significant protein aggregation for variants C3 and C4. It is concluded that the molecular design of the C-terminus in engineered ferritin, especially the E-helix, is important to ensure the epitope-based chimeric vaccine is safe (aggregate free) and efficacious.

An integrated and continuous downstream process for microbial virus-like particle vaccine biomanufacture.

In this study, we present the first integrated and continuous downstream process for the production of microbial virus-like particle vaccines. Modular murine polyomavirus major capsid VP1 with integrated J8 antigen was used as a model virus-like particle vaccine. The integrated continuous downstream process starts with crude cell lysate and consists of a flow-through chromatography step followed by periodic counter-current chromatography (PCC) (bind-elute) using salt-tolerant mixed-mode resin and subsequent in-line assembly. The automated process showed a robust behavior over different inlet feed concentrations ranging from 1.0 to 3.2 mg ml-1 with only minimal adjustments needed, and produced continuously high-quality virus-like particles, free of nucleic acids, with constant purity over extended periods of time. The average size remained constant between 44.8 ± 2.3 and 47.2 ± 2.9 nm comparable to literature. The process had an overall product recovery of 88.6% and a process productivity up to 2.56 mg h-1 mlresin-1 in the PCC step, depending on the inlet concentration. Integrating a flow through step with a subsequent PCC step allowed streamlined processing, showing a possible continuous pathway for a wide range of products of interest.

Control strategy for multi-column continuous periodic counter current chromatography subject to fluctuating inlet stream concentration.

Fluctuations of the inlet feed stream concentration are a challenge in controlling continuous multi-column counter current chromatography systems with standard methods. We propose a new control strategy based on calculated product column breakthrough from UV sensor signals by neglecting an impurity baseline and instead using the impurity to product ratio. This calculation is independent of the inlet feed concentration. In-silico simulation showed that the proposed method can calculate the product column breakthrough perfectly even with fluctuating and highly unstable inlet feed concentration during a loading cycle. Applying the proposed method to control a three column periodic counter current chromatography process with fluctuating inlet feed concentration resulted in constant column loading in each cycle, while using the standard method failed to do so. Unavoidable band broadening caused by diffusion and dispersion has been identified as an inherent limiting factor for accurate calculation of column breakthrough comparing inlet and outlet UV signals. The proposed advanced calculations increase the robustness of periodic counter current chromatography and extend the capability to process unstable inlet streams.

Immunogenicity study of engineered ferritins with C- and N-terminus insertion of Epstein-Barr nuclear antigen 1 epitope.

Human ferritin heavy chain, an example of a protein nanoparticle, has recently been used as a vaccine delivery platform. Human ferritin has advantages of uniform architecture, robust thermal and chemical stabilities, and good biocompatibility and biodegradation. There is however a lack of understanding about the relationship between insertion sites in ferritin (N-terminus and C-terminus) and the corresponding humoral and cell-mediated immune responses. To bridge this gap, we utilized an Epstein-Barr Nuclear Antigen 1 (EBNA1) epitope as a model to produce engineered ferritin-based vaccines E1F1 (N-terminus insertion) and F1E1 (C-terminus insertion) for the prevention of Epstein-Barr virus (EBV) infections. X-ray crystallography confirmed the relative positions of the N-terminus insertion and C-terminus insertion. For N-terminus insertion, the epitopes were located on the exterior surface of ferritin, while for C-terminus insertion, the epitopes were inside the ferritin cage. Based on the results of antigen-specific antibody titers from in-vivo tests, we found that there was no obvious difference on humoral immune responses between N-terminus and C-terminus insertion. We also evaluated splenocyte proliferation and memory lymphocyte T cell differentiation. Both results suggested C-terminus insertion produced a stronger proliferative response and cell-mediated immune response than N-terminus insertion. C-terminus insertion of EBNA1 epitope was also processed more efficiently by dendritic cells (DCs) than N-terminus insertion. This provides new insight into the relationship between the insertion site and immunogenicity of ferritin nanoparticle vaccines.

Stability of Engineered Ferritin Nanovaccines Investigated by Combined Molecular Simulation and Experiments.

Human ferritin is regarded as an attractive and promising vaccine platform because of its uniform structure, good plasticity, and desirable thermal and chemical stabilities. Besides, it is biocompatible and presumed safe when used as a vaccine carrier. However, there is a lack of knowledge of how different antigen insertion sites on the ferritin nanocage impact the resulting protein stability and performance. To address this question, we selected Epstein-Barr nuclear antigen 1 as a model epitope and fused it at the DNA level with different insertion sites, namely, the N- and C-termini of ferritin, to engineer proteins E1F1 and F1E1, respectively. Protein properties including hydrophobicity and thermal, pH, and chemical stability were investigated both by molecular dynamics (MD) simulation and by experiments. Both methods demonstrate that the insertion site plays an important role in protein properties. The C-terminus insertion (F1E1) leads to a less hydrophobic surface and more tolerance to the external influence of high temperature, pH, and high concentration of chemical denaturants compared to N-terminus insertion (E1F1). Simulated protein hydrophobicity and thermal stability by MD were in high accordance with experimental results. Thus, MD simulation can be used as a valuable tool to engineer nanovaccine candidates, cutting down costs by reducing the experimental effort and accelerating vaccine design.

Comparative evaluation of integrated purification pathways for bacterial modular polyomavirus major capsid protein VP1 to produce virus-like particles using high throughput process technologies.

Modular virus-like particles and capsomeres are potential vaccine candidates that can induce strong immune responses. There are many described protocols for the purification of microbially-produced viral protein in the literature, however, they suffer from inherent limitations in efficiency, scalability and overall process costs. In this study, we investigated alternative purification pathways to identify and optimise a suitable purification pathway to overcome some of the current challenges. Among the methods, the optimised purification strategy consists of an anion exchange step in flow through mode followed by a multi modal cation exchange step in bind and elute mode. This approach allows an integrated process without any buffer adjustment between the purification steps. The major contaminants like host cell proteins, DNA and aggregates can be efficiently removed by the optimised strategy, without the need for a size exclusion polishing chromatography step, which otherwise could complicate the process scalability and increase overall cost. High throughput process technology studies were conducted to optimise binding and elution conditions for multi modal cation exchanger, Capto™ MMC and strong anion exchanger Capto™ Q. A dynamic binding capacity of 14 mg ml-1 was achieved for Capto™ MMC resin. Samples derived from each purification process were thoroughly characterized by RP-HPLC, SEC-HPLC, SDS-PAGE and LC-ESI-MS/MS Mass Spectrometry analytical methods. Modular polyomavirus major capsid protein could be purified within hours using the optimised process achieving purities above 87% and above 96% with inclusion of an initial precipitation step. Purified capsid protein could be easily assembled in-vitro into well-defined virus-like particles by lowering pH with addition of calcium chloride to the eluate. High throughout studies allowed the screening of a vast design space within weeks, rather than months, and unveiled complicated binding behaviour for CaptoTM MMC.

Virus-like particle preparation is improved by control over capsomere-DNA interactions during chromatographic purification.

Expression of viral capsomeres in bacterial systems and subsequent in vitro assembly into virus-like particles is a possible pathway for affordable future vaccines. However, purification is challenging as viral capsomeres show poor binding to chromatography media. In this study, the behavior of capsomeres in unfractionated bacterial lysate was compared with that for purified capsomeres, with or without added microbial DNA, to better understand reasons for poor bioprocess behavior. We show that aggregates or complexes form through the interaction between viral capsomeres and DNA, especially in bacterial lysates rich in contaminating DNA. The formation of these complexes prevents the target protein capsomeres from accessing the pores of chromatography media. We find that protein-DNA interactions can be modulated by controlling the ionic strength of the buffer and that at elevated ionic strengths the protein-DNA complexes dissociate. Capsomeres thus released show enhanced bind-elute behavior on salt-tolerant chromatography media. DNA could therefore be efficiently removed. We believe this is the first report of the use of an optimized salt concentration that dissociates capsomere-DNA complexes yet enables binding to salt-tolerant media. Post purification, assembly experiments indicate that DNA-protein interactions can play a negative role during in vitro assembly, as DNA-protein complexes could not be assembled into virus-like particles, but formed worm-like structures. This study reveals that the control over DNA-protein interaction is a critical consideration during downstream process development for viral vaccines.

Immunogenicity and Vaccine Efficacy Boosted by Engineering Human Heavy Chain Ferritin and Chimeric Hepatitis B Virus Core Nanoparticles.

Human heavy-chain ferritin (HFn) and hepatitis B virus core (HBc) are both nanoparticle proteins presenting a well-oriented architecture with constant size and shape, which can be engineered to carry epitopes on the surface of the nanoparticle protein cage, enabling vaccine design. This study aims to investigate the immunogenicity differences between engineered HFn and chimeric HBc bearing the same epitope. As a proof of concept, the model epitope Epstein-Barr nuclear antigen 1 (EBNA1) is inserted at the N-terminus of the HFn and HBc subunit to produce two vaccine candidates named EBNA1-HFn (E1F1) and EBNA1-HBc (E1H1), respectively. From in vivo immunogenicity studies, E1H1 demonstrates the capability to prompt significant humoral and cell-mediated immune responses in adjuvant-free formulation. When formulated with the aluminum hydroxide adjuvant, E1H1 produces approximately 5× higher titer and 2× stronger proliferation index (PI) than E1F1. These results confirm that the HBc carrier induces a stronger humoral immune response than HFn. On the other hand, from lymphocyte activation experiments, E1F1 induces a stronger cell-mediated immune response indicated by 5× more CD8+T cells and 2× more effector memory T cells in the E1F1 group versus the E1H1 group. Through this study, HFn and HBc are shown to be potentially effective vaccine carrier nanoparticles having subtly different immunological responses.

Chimeric Virus-Like Particles and Capsomeres Induce Similar CD8+ T Cell Responses but Differ in Capacity to Induce CD4+ T Cell Responses and Antibody Responses.

Despite extensive research, the development of an effective malaria vaccine remains elusive. The induction of robust and sustained T cell and antibody response by vaccination is an urgent unmet need. Chimeric virus-like particles (VLPs) are a promising vaccine platform. VLPs are composed of multiple subunit capsomeres which can be rapidly produced in a cost-effective manner, but the ability of capsomeres to induce antigen-specific cellular immune responses has not been thoroughly investigated. Accordingly, we have compared chimeric VLPs and their sub-unit capsomeres for capacity to induce CD8+ and CD4+ T cell and antibody responses. We produced chimeric murine polyomavirus VLPs and capsomeres each incorporating defined CD8+ T cell, CD4+ T cell or B cell repeat epitopes derived from Plasmodium yoelii CSP. VLPs and capsomeres were evaluated using both homologous or heterologous DNA prime/boost immunization regimens for T cell and antibody immunogenicity. Chimeric VLP and capsomere vaccine platforms induced robust CD8+ T cell responses at similar levels which was enhanced by a heterologous DNA prime. The capsomere platform was, however, more efficient at inducing CD4+ T cell responses and less efficient at inducing antigen-specific antibody responses. Our data suggest that capsomeres, which have significant manufacturing advantages over VLPs, should be considered for diseases where a T cell response is the desired outcome.

Nanoparticle elasticity regulates phagocytosis and cancer cell uptake.

The ability of cells to sense external mechanical cues is essential for their adaptation to the surrounding microenvironment. However, how nanoparticle mechanical properties affect cell-nanoparticle interactions remains largely unknown. Here, we synthesized a library of silica nanocapsules (SNCs) with a wide range of elasticity (Young's modulus ranging from 560 kPa to 1.18 GPa), demonstrating the impact of SNC elasticity on SNC interactions with cells. Transmission electron microscopy revealed that the stiff SNCs remained spherical during cellular uptake. The soft SNCs, however, were deformed by forces originating from the specific ligand-receptor interaction and membrane wrapping, which reduced their cellular binding and endocytosis rate. This work demonstrates the crucial role of the elasticity of nanoparticles in modulating their macrophage uptake and receptor-mediated cancer cell uptake, which may shed light on the design of drug delivery vectors with higher efficiency.

Impact of Site-Specific Bioconjugation on the Interfacial Activity of a Protein Biosurfactant.

Biosurfactants are surface active molecules that can be produced by renewable, industrially scalable biologic processes. DAMP4, a designer biosurfactant, enables the modification of interfaces via genetic or chemical fusion to functional moieties. However, bioconjugation of addressable amines introduces heterogeneity that limits the precision of functionalization as well as the resolution of interfacial characterization. Here, we designed DAMP4 variants with cysteine point mutations to allow for site-specific bioconjugation. The DAMP4 variants were shown to retain the structural stability and interfacial activity characteristic of the parent molecule, while permitting efficient and specific conjugation of polyethylene glycol (PEG). PEGylation results in a considerable reduction on the interfacial activity of both single and double mutants. Comparison of conjugates with one or two conjugation sites shows that both the number of conjugates as well as the mass of conjugated material impact the interfacial activity of DAMP4. As a result, the ability of DAMP4 variants with multiple PEG conjugates to impart colloidal stability on peptide-stabilized emulsions is reduced. We suggest that this is due to steric constraints on the structures of amphiphilic helices at the interface. Specific and efficient bioconjugation permits the exploration and investigation of the interfacial properties of designer protein biosurfactants with molecular precision. Our findings should therefore inform the design and modification of biosurfactants for their increasing use in industrial processes and nutritional and pharmaceutical formulations.

Evaluation of baiting fipronil-loaded silica nanocapsules against termite colonies in fields.

Various pesticide nanocarriers have been developed. However, their pest-control applications remain limited in laboratories. Herein, we developed silica nanocapsules encapsulating fipronil (SNC) and their engineered form, poly(ethyleneimine)-coated SNC (SNC-PEI), based on recombinant catalytic modular protein D4S2 and used them against termite colonies Coptotermes lacteus in fields. To achieve this, an integrated biomolecular bioprocess was developed to produce D4S2 for manufacturing SNC containing fipronil with high encapsulation efficiency of approximately 97% at benign reaction conditions and at scales sufficient for the field applications. PEI coating was achieved via electrostatic interactions to yield SNC-PEI with a slower release of fipronil than SNC without coating. As a proof-of-concept, bait toxicants containing varied fipronil concentrations were formulated and exposed to nine termite mounds, aiming to prolong fipronil release hence allowing sufficient time for termites to relocate the baits into and distribute throughout the colony, and to eliminate that colony. Some baits were relocated into the mounds, but colonies were not eliminated due to several reasons. We caution others interested in producing bait toxicants to be aware of the multilevel resistance mechanisms of the Coptotermes spp. "superorganism".

Bioinspired Core-Shell Nanoparticles for Hydrophobic Drug Delivery.

A large range of nanoparticles have been developed to encapsulate hydrophobic drugs. However, drug loading is usually less than 10 % or even 1 %. Now, core-shell nanoparticles are fabricated having exceptionally high drug loading up to 65 % (drug weight/the total weight of drug-loaded nanoparticles) and high encapsulation efficiencies (>99 %) based on modular biomolecule templating. Bifunctional amphiphilic peptides are designed to not only stabilize hydrophobic drug nanoparticles but also induce biosilicification at the nanodrug particle surface thus forming drug-core silica-shell nanocomposites. This platform technology is highly versatile for encapsulating various hydrophobic cargos. Furthermore, the high drug loading nanoparticles lead to better in vitro cytotoxic effects and in vivo suppression of tumor growth, highlighting the significance of using high drug-loading nanoparticles.

Chimeric Murine Polyomavirus Virus-Like Particles Induce Plasmodium Antigen-Specific CD8+ T Cell and Antibody Responses.

An effective vaccine against the Plasmodium parasite is likely to require the induction of robust antibody and T cell responses. Chimeric virus-like particles are an effective vaccine platform for induction of antibody responses, but their capacity to induce robust cellular responses and cell-mediated protection against pathogen challenge has not been established. To evaluate this, we produced chimeric constructs using the murine polyomavirus structural protein with surface-exposed CD8+ or CD4+ T cell or B cell repeat epitopes derived from the Plasmodium yoelii circumsporozoite protein, and assessed immunogenicity and protective capacity in a murine model. Robust CD8+ T cell responses were induced by immunization with the chimeric CD8+ T cell epitope virus-like particles, however CD4+ T cell responses were very low. The B cell chimeric construct induced robust antibody responses but there was no apparent synergy when T cell and B cell constructs were administered as a pool. A heterologous prime/boost regimen using plasmid DNA priming followed by a VLP boost was more effective than homologous VLP immunization for cellular immunity and protection. These data show that chimeric murine polyomavirus virus-like particles are a good platform for induction of CD8+ T cell responses as well as antibody responses.

Synthetic biology for bioengineering virus-like particle vaccines.

Vaccination is the most effective method of disease prevention and control. Many viruses and bacteria that once caused catastrophic pandemics (e.g., smallpox, poliomyelitis, measles, and diphtheria) are either eradicated or effectively controlled through routine vaccination programs. Nonetheless, vaccine manufacturing remains incredibly challenging. Viruses exhibiting high antigenic diversity and high mutation rates cannot be fairly contested using traditional vaccine production methods and complexities surrounding the manufacturing processes, which impose significant limitations. Virus-like particles (VLPs) are recombinantly produced viral structures that exhibit immunoprotective traits of native viruses but are noninfectious. Several VLPs that compositionally match a given natural virus have been developed and licensed as vaccines. Expansively, a plethora of studies now confirms that VLPs can be designed to safely present heterologous antigens from a variety of pathogens unrelated to the chosen carrier VLPs. Owing to this design versatility, VLPs offer technological opportunities to modernize vaccine supply and disease response through rational bioengineering. These opportunities are greatly enhanced with the application of synthetic biology, the redesign and construction of novel biological entities. This review outlines how synthetic biology is currently applied to engineer VLP functions and manufacturing process. Current and developing technologies for the identification of novel target-specific antigens and their usefulness for rational engineering of VLP functions (e.g., presentation of structurally diverse antigens, enhanced antigen immunogenicity, and improved vaccine stability) are described. When applied to manufacturing processes, synthetic biology approaches can also overcome specific challenges in VLP vaccine production. Finally, we address several challenges and benefits associated with the translation of VLP vaccine development into the industry.

Synergetic Combinations of Dual-Targeting Ligands for Enhanced In Vitro and In Vivo Tumor Targeting.

The concept of dual-ligand targeting has been around for quite some time, but remains controversial due to the intricate interplay between so many different factors such as the choice of dual ligands, their densities, ratios and length matching, etc. Herein, the synthesis of a combinatorial library of single and dual-ligand nanoparticles with systematically varied properties (ligand densities, ligand ratios, and lengths) for tumor targeting is reported. Folic acid (FA) and hyaluronic acid (HA) are used as two model targeting ligands. It is found that the length matching and ligand ratio play critical roles in achieving the synergetic effect of the dual-ligand targeting. When FA is presented on the nanoparticle surface through a 5K polyethylene glycol (PEG) chain, the dual ligand formulations using the HA with either 5K or 10K length do not show any targeting effect, but the right length of HA (7K) with a careful selection of the right ligand ratio do enhance the targeting efficiency and specificity significantly. Further in vitro 3D tumor spheroid models and in vivo xenograft mice models confirm the synergetic targeting efficiency of the optimal dual-ligand formulation (5F2H7K ). This work provides a valuable insight into the design of dual-ligand targeting nanosystems.

Understanding the Effects of Nanocapsular Mechanical Property on Passive and Active Tumor Targeting.

The physicochemical properties of nanoparticles (size, charge, and surface chemistry, etc.) influence their biological functions often in complex and poorly understood ways. This complexity is compounded when the nanostructures involved have variable mechanical properties. Here, we report the synthesis of liquid-filled silica nanocapsules (SNCs, ∼ 150 nm) having a wide range of stiffness (with Young's moduli ranging from 704 kPa to 9.7 GPa). We demonstrate a complex trade-off between nanoparticle stiffness and the efficiencies of both immune evasion and passive/active tumor targeting. Soft SNCs showed 3 times less uptake by macrophages than stiff SNCs, while the uptake of PEGylated SNCs by cancer cells was independent of stiffness. In addition, the functionalization of stiff SNCs with folic acid significantly enhanced their receptor-mediated cellular uptake, whereas little improvement for the soft SNCs was conferred. Further in vivo experiments confirmed these findings and demonstrated the critical role of nanoparticle mechanical properties in regulating their interactions with biological systems.

Self-adjuvanting nanoemulsion targeting dendritic cell receptor Clec9A enables antigen-specific immunotherapy.

Non-antigen-specific stimulatory cancer immunotherapies are commonly complicated by off-target effects. Antigen-specific immunotherapy, combining viral tumor antigen or personalized neoepitopes with immune targeting, offers a solution. However, the lack of flexible systems targeting tumor antigens to cross-presenting dendritic cells (DCs) limits clinical development. Although antigen-anti-Clec9A mAb conjugates target cross-presenting DCs, adjuvant must be codelivered for cytotoxic T lymphocyte (CTL) induction. We functionalized tailored nanoemulsions encapsulating tumor antigens to target Clec9A (Clec9A-TNE). Clec9A-TNE encapsulating OVA antigen targeted and activated cross-presenting DCs without additional adjuvant, promoting antigen-specific CD4+ and CD8+ T cell proliferation and CTL and antibody responses. OVA-Clec9A-TNE-induced DC activation required CD4 and CD8 epitopes, CD40, and IFN-α. Clec9A-TNE encapsulating HPV E6/E7 significantly suppressed HPV-associated tumor growth, while E6/E7-CpG did not. Clec9A-TNE loaded with pooled B16-F10 melanoma neoepitopes induced epitope-specific CD4+ and CD8+ T cell responses, permitting selection of immunogenic neoepitopes. Clec9A-TNE encapsulating 6 neoepitopes significantly suppressed B16-F10 melanoma growth in a CD4+ T cell-dependent manner. Thus, cross-presenting DCs targeted with antigen-Clec9A-TNE stimulate therapeutically effective tumor-specific immunity, dependent on T cell help.