With the ever-present threat of pandemics, it is imperative vaccine technologies eliciting broad and durable immunity to high-risk pathogens are developed. Yet, current annual influenza vaccines, for example, fail to provide robust immunity against the 3-4 homologous strains they contain, let alone heterologous strains. Herein, this study demonstrates that sustained delivery of multivalent influenza vaccines from an injectable polymer-nanoparticle (PNP) hydrogel technology induces more rapid, consistent, and potent humoral immune responses against multiple homologous viruses, as well as potent responses against heterologous viruses and potential pandemic subtypes H5N1, H7N9 and H9N2. Further, admixing PNP hydrogels with commercial influenza vaccines results in stronger hemagglutination inhibition against both heterologous and homologous viruses. Additional investigation shows this enhanced potency and breadth arise from higher affinity antibodies targeting both the hemagglutinin stem and head. Overall, this simple and effective sustained delivery platform for multivalent annual influenza vaccines generates durable, potent, and remarkably broad immunity to influenza.

Hydrogels have emerged as promising materials for bioprinting and many other biomedical applications due to their high degree of biocompatibility and ability to support and/or modulate cell viability and function. Yet, many hydrogel bioinks have suffered from low efficiency due to limitations on accessible printing speeds, often limiting cell viability and/or the constructs which can be generated. In this study, a highly extensible bioink system created by modulating the rheology of physically crosslinked hydrogels comprising hydrophobically-modified cellulosics and either surfactants or cyclodextrins is reported. It is demonstrated that these hydrogels are highly shear-thinning with broadly tunable viscoelasticity and stress-relaxation through simple modulation of the composition. Rheological experiments demonstrate that increasing concentration of rheology-modifying additives yields hydrogel materials exhibiting extensional strain-to-break values up to 2000%, which is amongst the most extensible examples of physically crosslinked hydrogels of this type. The potential of these hydrogels for use as bioinks is demonstrated by evaluating the relationship between extensibility and printability, demonstrating that greater hydrogel extensibility enables faster print speeds and smaller print features. The findings suggest that optimizing hydrogel extensibility can enhance high-speed 3D bioprinting capabilities, reporting over 5000 fold enhancement in speed index compared to existing works reported for hydrogel-based bioinks in extrusion-based printing.

Messenger RNA (mRNA) delivered in lipid nanoparticles (LNPs) rose to the forefront of vaccine candidates during the COVID-19 pandemic due to scalability, adaptability, and potency. Yet, there remain critical areas for improvements of these vaccines in durability and breadth of humoral responses. In this work, we explore a modular strategy to target mRNA/LNPs to antigen-presenting cells with an injectable polymer-nanoparticle (PNP) hydrogel technology, which recruits key immune cells and forms an immunological niche in vivo. We characterize this niche on a single-cell level and find it is highly tunable through incorporation of adjuvants like MPLAs and 3M-052. Delivering commercially available severe acute respiratory syndrome coronavirus 2 mRNA vaccines in PNP hydrogels improves the durability and quality of germinal center reactions, and the magnitude, breadth, and durability of humoral responses. The tunable immune niche formed within PNP hydrogels effectively skews immune responses based on encapsulated adjuvants, creating opportunities to precisely modulate mRNA/LNP vaccines for various indications from infectious diseases to cancers.

Over the past few decades, the development of potent and safe immune-activating adjuvant technologies has become the heart of intensive research in the constant fight against highly mutative and immune evasive viruses such as influenza, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and human immunodeficiency virus (HIV). Herein, we developed a highly modular saponin-based nanoparticle platform incorporating Toll-like receptor agonists (TLRas) including TLR1/2a, TLR4a, and TLR7/8a adjuvants and their mixtures. These various TLRa–saponin nanoparticle adjuvant constructs induce unique acute cytokine and immune-signaling profiles, leading to specific T helper responses that could be of interest depending on the target disease for prevention. In a murine vaccine study, the adjuvants greatly improved the potency, durability, breadth, and neutralization of both COVID-19 and HIV vaccine candidates, suggesting the potential broad application of these adjuvant constructs to a range of different antigens. Overall, this work demonstrates a modular TLRa-SNP adjuvant platform that could improve the design of vaccines and affect modern vaccine development.

Development of effective vaccines for infectious diseases has been one of the most successful global health interventions in history. Though, while ideal subunit vaccines strongly rely on antigen and adjuvant(s) selection, the mode and time scale of exposure to the immune system has often been overlooked. Unfortunately, poor control over the delivery of many adjuvants, which play a key role in enhancing the quality and potency of immune responses, can limit their efficacy and cause off-target toxicities. There is a critical need for improved adjuvant delivery technologies to enhance their efficacy and boost vaccine performance. Nanoparticles have been shown to be ideal carriers for improving antigen delivery due to their shape and size, which mimic viral structures but have been generally less explored for adjuvant delivery. Here, we describe the design of self-assembled poly(ethylene glycol)-b-poly(lactic acid) nanoparticles decorated with CpG, a potent TLR9 agonist, to increase adjuvanticity in COVID-19 vaccines. By controlling the surface density of CpG, we show that intermediate valency is a key factor for TLR9 activation of immune cells. When delivered with the SARS-CoV-2 spike protein, CpG nanoparticle (CpG-NP) adjuvant greatly improves the magnitude and duration of antibody responses when compared to soluble CpG, and results in overall greater breadth of immunity against variants of concern. Moreover, encapsulation of CpG-NP into injectable polymeric-nanoparticle (PNP) hydrogels enhances the spatiotemporal control over codelivery of CpG-NP adjuvant and spike protein antigen such that a single immunization of hydrogel-based vaccines generates humoral responses comparable to those of a typical prime-boost regimen of soluble vaccines. These delivery technologies can potentially reduce the costs and burden of clinical vaccination, both of which are key elements in fighting a pandemic.

Viruses pose a significant threat to both global health and the global economy. It is clear that novel antiviral strategies are urgently needed, with a broad-spectrum approach being most desired. We have discovered a broad-spectrum, non-toxic polymer virucide that can tackle the viral threat. This polymeric virucide is effective at nanomolar concentrations, against a broad-spectrum of viruses and, demonstrated using an intranasal respiratory syncytial virus (RSV) murine model, has excellent efficacy, low anti-coagulant properties and low toxicity in vivo . Molecular dynamic simulations show that this polymer achieves its virucidal antiviral effect via self-assembly of viral-receptors leading to increased envelope forces and viral disassembly. The discovery of this cheap and readily produced polymer marks the start of a new type of receptor-crosslinking broad-spectrum antiviral that has significant potential to combat the global threat posed by viruses. ### Competing Interest Statement The authors have declared no competing interest.

Most vaccines require several immunizations to induce robust immunity, and indeed, most SARS-CoV-2 vaccines require an initial two-shot regimen followed by several boosters to maintain efficacy. Such a complex series of immunizations unfortunately increases the cost and complexity of populations-scale vaccination and reduces overall compliance and vaccination rate. In a rapidly evolving pandemic affected by the spread of immune-escaping variants, there is an urgent need to develop vaccines capable of providing robust and durable immunity. In this work, a single immunization SARS-CoV-2 subunit vaccine is developed that can rapidly generate potent, broad, and durable humoral immunity. Injectable polymer-nanoparticle (PNP) hydrogels are leveraged as a depot technology for the sustained delivery of a nanoparticle antigen (RND-NP) displaying multiple copies of the SARS-CoV-2 receptor-binding domain (RBD) and potent adjuvants including CpG and 3M-052. Compared to a clinically relevant prime-boost regimen with soluble vaccines formulated with CpG/alum or 3M-052/alum adjuvants, PNP hydrogel vaccines more rapidly generated higher, broader, and more durable antibody responses. Additionally, these single-immunization hydrogel-based vaccines elicit potent and consistent neutralizing responses. Overall, it is shown that PNP hydrogels elicit improved anti-COVID immune responses with only a single administration, demonstrating their potential as critical technologies to enhance overall pandemic readiness.

Equitable global access to vaccines requires we overcome challenges associated with complex immunization schedules and their associated economic burdens that hinder delivery in under resourced environments. The rabies vaccine, for example, requires multiple immunizations for effective protection and each dose is cost prohibitive, and therefore inaccessibility disproportionately impacts low- and middle-income countries. In this work we developed an injectable hydrogel depot technology for sustained delivery of commercial inactivated rabies virus vaccines. In a mouse model, we showed that a single immunization of a hydrogel-based rabies vaccine elicited comparable antibody titers to a standard prime-boost bolus regimen of a commercial rabies vaccine, despite these hydrogel vaccines comprising only half of the total dose delivered in the bolus control. Moreover, these hydrogel-based vaccines elicited similar antigen-specific T-cell responses and neutralizing antibody responses compared to the bolus vaccine. Notably, we demonstrated that while addition of a potent clinical TLR4 agonist adjuvant to the gels slightly improved binding antibody responses, inclusion of this adjuvant to the inactivated virion vaccine was detrimental to neutralizing responses. Taken together, these results suggest that these hydrogels can enable an effective regimen compression and dosesparing strategy for improving global access to vaccines.

Development of effective vaccines for infectious diseases has been one of the most successful global health interventions in history. Though, while ideal subunit vaccines strongly rely on antigen and adjuvant(s) selection, the mode and timescale of exposure to the immune system has often been overlooked. Unfortunately, poor control over the delivery of many adjuvants, which play a key role in enhancing the quality and potency of immune responses, can limit their efficacy and cause off-target toxicities. There is critical need for new adjuvant delivery technologies to enhance their efficacy and boost vaccine performance. Nanoparticles have been shown to be ideal carriers for improving antigen delivery due to their shape and size, which mimic viral structures, but have been generally less explored for adjuvant delivery. Here, we describe the design of self-assembled poly(ethylene glycol)-b-poly(lactic acid) nanoparticles decorated with CpG, a potent TLR9 agonist, to increase adjuvanticity in COVID-19 vaccines. By controlling the surface density of CpG, we show that intermediate valency is a key factor for TLR9 activation of immune cells. When delivered with the SARS-CoV-2 spike protein, CpG nanoparticle (CpG-NP) adjuvant greatly improve the magnitude and duration of antibody responses when compared to soluble CpG, and result in overall greater breadth of immunity against variants of concern. Moreover, encapsulation of CpG-NP into injectable polymeric-nanoparticle (PNP) hydrogels enhance the spatiotemporal control over co-delivery of CpG-NP adjuvant and spike protein antigen such that a single immunization of hydrogel-based vaccines generates comparable humoral responses as a typical prime-boost regimen of soluble vaccines. These delivery technologies can potentially reduce the costs and burden of clinical vaccination, both of which are key elements in fighting a pandemic.

Hydrogels that can be injected into the body using standard needles or catheters enable a minimally invasive strategy to prolong local delivery of therapeutic drug and cellular cargo. In particular, physically crosslinked hydrogels exhibit shear-thinning and self-healing behaviors enabling facile injectability and depot formation upon administration. While prior efforts to characterize these systems have focused on injectability and cargo release behaviors, prediction of cargo release in the body often assumes the materials form a depot rather than spreading out upon administration. Here, we evaluate how hydrogel rheology correlates with depot formation and persistence following subcutaneous administration in mice with two physicochemically-distinct, physically crosslinked hydrogel systems. We evaluate calcium-alginate and polymer-nanoparticle hydrogel systems exhibiting variable mechanical behaviors across several rheological properties (stiffness, viscoelasticity, yield stress, and creep). By relating measured rheological properties to depot formation and persistence time following subcutaneous administration, we identify that yield stress is predictive of initial depot formation while creep is predictive of depot persistence. Indeed, only materials with yield stresses greater than 25 Pa form robust depots and reduced creep correlates with longer depot persistence. These findings provide predictive insights into design considerations for hydrogel technologies capable of extended controlled release of therapeutic cargo.

The immune system is one of the most important, complex biological networks regulating and protecting human health. Its precise modulation can prevent deadly infections and fight cancer. Accordingly, prophylactic vaccines and cancer immunotherapies are some of the most powerful technologies to protect against potential dangers through training of the immune system. Upon immunization, activation and maturation of B and T cells of the adaptive immune system are necessary for development of proper humoral and cellular protection. Yet, the exquisite organization of the immune system requires spatiotemporal control over the exposure of immunomodulatory signals. For example, while the human immune system has evolved to develop immunity to natural pathogenic infections that often last for weeks, current prophylactic vaccination technologies only expose the immune system to immunomodulatory signals for hours to days. It has become clear that leveraging sustained release technologies to prolong immunogen and adjuvant exposure can increase the potency, durability, and quality of adaptive immune responses. Over the past several years, tremendous breakthroughs have been made in the design of novel biomaterials such as nanoparticles, microparticles, hydrogels, and microneedles that can precisely control the presentation of immunomodulatory signals to the immune system. In this review, we discuss relevant sustained release strategies and their corresponding benefits to cellular and humoral responses.

When properly deployed, the immune system can eliminate deadly pathogens, eradicate metastatic cancers, and provide long-lasting protection from diverse diseases. Unfortunately, realizing these remarkable capabilities is inherently risky as disruption to immune homeostasis can elicit dangerous complications or autoimmune disorders. While current research is continuously expanding the arsenal of potent immunotherapeutics, there is a technological gap when it comes to controlling when, where, and how long these drugs act on the body. Here, this study explored the ability of a slow-releasing injectable hydrogel depot to reduce dose-limiting toxicities of immunostimulatory CD40 agonist (CD40a) while maintaining its potent anticancer efficacy. A previously described polymer-nanoparticle (PNP) hydrogel system is leveraged that exhibits shear-thinning and yield-stress properties that are hypothesized to improve locoregional delivery of CD40a immunotherapy. Using positron emission tomography, it is demonstrated that prolonged hydrogel-based delivery redistributes CD40a exposure to the tumor and the tumor draining lymph node (TdLN), thereby reducing weight loss, hepatotoxicity, and cytokine storm associated with standard treatment. Moreover, CD40a-loaded hydrogels mediate improved local cytokine induction in the TdLN and improve treatment efficacy in the B16F10 melanoma model. PNP hydrogels, therefore, represent a facile, drug-agnostic method to ameliorate immune-related adverse effects and explore locoregional delivery of immunostimulatory drugs.

The sustained release of vaccine cargo has been shown to improve humoral immune responses to challenging pathogens such as influenza. Extended codelivery of antigen and adjuvant prolongs germinal center reactions, thus improving antibody affinity maturation and the ability to neutralize the target pathogen. Here, we develop an injectable, physically cross-linked polymer-nanoparticle (PNP) hydrogel system to prolong the local codelivery of hemagglutinin and a toll-like receptor 7/8 agonist (TLR7/8a) adjuvant. By tethering the TLR7/8a to a NP motif within the hydrogels (TLR7/8a-NP), the dynamic mesh of the PNP hydrogels enables codiffusion of the adjuvant and protein antigen (hemagglutinin), therefore enabling sustained codelivery of these two physicochemically distinct molecules. We show that subcutaneous delivery of PNP hydrogels carrying hemagglutinin and TLR7/8a-NP in mice improves the magnitude and duration of antibody titers in response to a single injection vaccination compared to clinically used adjuvants. Furthermore, the PNP gel-based slow delivery of influenza vaccines led to increased breadth of antibody responses against future influenza variants, including a future pandemic variant, compared to clinical adjuvants. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of influenza subunit vaccines.

Vaccines are critical for combating infectious diseases across the globe. Influenza, for example, kills roughly 500,000 people annually worldwide, despite annual vaccination campaigns. Efficacious vaccines must elicit a robust and durable antibody response, and poor efficacy often arises from inappropriate temporal control over antigen and adjuvant presentation to the immune system. In this work, we sought to exploit the immune system's natural response to extended pathogen exposure during infection by designing an easily administered slow-delivery influenza vaccine platform. We utilized an injectable and self-healing polymer-nanoparticle (PNP) hydrogel platform to prolong the co-delivery of vaccine components to the immune system. We demonstrated that these hydrogels exhibit unique dynamic physical characteristics whereby physicochemically distinct influenza hemagglutinin antigen and a toll-like receptor 7/8 agonist adjuvant could be co-delivered over prolonged timeframes that were tunable through simple alteration of the gel formulation. We show a relationship between hydrogel physical properties and the resulting immune response to immunization. When administered in mice, hydrogel-based vaccines demonstrated enhancements in the magnitude and duration of humoral immune responses compared to alum, a widely used clinical adjuvant system. We found stiffer hydrogel formulations exhibited slower release and resulted in the greatest improvements to the antibody response while also enabling significant adjuvant dose sparing. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of influenza subunit vaccines.

Understanding structural transitions within macromolecules remains an important challenge in biochemistry, with important implications for drug development and medicine. Insight into molecular behavior often requires residue-specific dynamics measurement at micromolar concentrations. We studied MP01-Gen4, a library peptide selected to rapidly undergo bioconjugation, by using electron paramagnetic resonance (EPR) to measure conformational dynamics. We mapped the dynamics of MP01-Gen4 with residue-specificity and identified the regions involved in a structural transformation related to the conjugation reaction. Upon reaction, the conformational dynamics of residues near the termini slow significantly more than central residues, indicating that the reaction induces a structural transition far from the reaction site. Arrhenius analysis demonstrates a nearly threefold decrease in the activation energy of conformational diffusion upon reaction (8.0 k B T to 3.4 k B T ), which occurs across the entire peptide, independently of residue position. This novel approach to EPR spectral analysis provides insight into the positional extent of disorder and the nature of the energy landscape of a highly reactive, intrinsically disordered library peptide before and after conjugation.

We investigate the fabrication of sub-20 nm pillars by DSA lithography using PS-cylinder-forming PS-b-PMMA block copolymer (BCP). The approach is based on the removal of PMMA-matrix by either dry or wet etching to form PS pillars which act as a soft etching mask that can be further transferred to an intermediate hard one and then to the substrate. The process conditions of BCP self-assembly were optimized in terms of annealing temperature, brush layer composition and film thickness. It was demonstrated that PS/PMMA volume fractions of 50/50 in the PS-r-PMMA brush layer is the most adapted to obtain standing PS cylinders. Top-down SEM images showed a hexagonal array of PS cylinders in a PMMA matrix with a natural period of 33.5 nm (determined by Fast Fourier Transform FFT method) and CD around 15 nm. Both wet and dry etching strategies for PMMA removal were discussed. It was shown that UV exposure followed by organic solvent development is necessary to decrease pattern collapsing during PMMA removal step which is the most critical issue for pillar fabrication. PMMA removal by dry etching was shown to completely avoid this pattern collapsing. Finally, pillars etching transfer to typical organic Si containing antireflective coating and spin on carbon (SiARC/SOC) hard mask and then to the silicon substrate was demonstrated. Si pillars of 15 nm CD and 70 nm height were obtained with a straight profile shape.

Simpler and more rapid approaches for therapeutic drug-level monitoring are highly desirable to enable use at the point-of-care. We have developed an all-electronic approach for detection of the HIV drug tenofovir based on scalable fabrication of arrays of graphene field-effect transistors (GFETs) functionalized with a commercially available DNA aptamer. The shift in the Dirac voltage of the GFETs varied systematically with the concentration of tenofovir in deionized water, with a detection limit less than 1 ng/mL. Tests against a set of negative controls confirmed the specificity of the sensor response. This approach offers the potential for further development into a rapid and convenient point-of-care tool with clinically relevant performance.