Nanoparticle-Conjugated Toll-Like Receptor 9 Agonists Improve the Potency, Durability, and Breadth of COVID-19 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 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.

Soft Viscoelastic Magnetic Hydrogels from the In Situ Mineralization of Iron Oxide in Metal-Coordinate Polymer Networks.

The design of soft magnetic hydrogels with high concentrations of magnetic particles is complicated by weak retention of the iron oxide particles in the hydrogel scaffold. Here, we propose a design strategy that circumvents this problem through the in situ mineralization of iron oxide nanoparticles within polymer hydrogels functionalized with strongly iron-coordinating nitrocatechol groups. The mineralization process facilitates the synthesis of a high concentration of large iron oxide nanoparticles (up to 57 wt % dry mass per single cycle) in a simple one-step process under ambient conditions. The resulting hydrogels are soft (kPa range) and viscoelastic and exhibit strong magnetic actuation. This strategy offers a pathway for the energy-efficient design of soft, mechanically robust, and magneto-responsive hydrogels for biomedical applications.

Saponin Nanoparticle Adjuvants Incorporating Toll-Like Receptor Agonists Improve Vaccine Immunomodulation.

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, SARS-CoV-2, and HIV. Herein, we developed a highly modular saponin-based nanoparticle platform incorporating toll-like receptor agonists (TLRas) including TLR1/2a, TLR4a, TLR7/8a adjuvants and their mixtures. These various TLRa-SNP adjuvant constructs induce unique acute cytokine and immune-signaling profiles, leading to specific Th-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 which could improve the design of vaccines for and dramatically impact modern vaccine development.

Broad and Durable Humoral Responses Following Single Hydrogel Immunization of SARS-CoV-2 Subunit Vaccine.

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.

A regimen compression strategy for commercial vaccines leveraging an injectable hydrogel depot technology for sustained vaccine exposure.

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.

Injectable Nanoparticle-Based Hydrogels Enable the Safe and Effective Deployment of Immunostimulatory CD40 Agonist Antibodies.

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.

Sustained delivery approaches to improving adaptive immune responses.

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.

Modulation of injectable hydrogel properties for slow co-delivery of influenza subunit vaccine components enhance the potency of humoral immunity.

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.

Prolonged Codelivery of Hemagglutinin and a TLR7/8 Agonist in a Supramolecular Polymer-Nanoparticle Hydrogel Enhances Potency and Breadth of Influenza Vaccination.

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.

Gelation and yielding behavior of polymer-nanoparticle hydrogels.

Polymer-nanoparticle hydrogels are a unique class of self-assembled, shear-thinning, yield-stress fluids that have demonstrated potential utility in many impactful applications. Here, we present a thorough analysis of the gelation and yielding behavior of these materials with respect to the polymer and nanoparticle component stoichiometry. Through comprehensive rheological and diffusion studies, we reveal insights into the structural dynamics of the polymer nanoparticle network that identify that stoichiometry plays a key role in gelation and yielding, ultimately enabling the development of hydrogel formulations with unique shear-thinning and yield-stress behaviors. Access to these materials opens new doors for interesting applications in a variety of fields including tissue engineering, drug delivery, and controlled solution viscosity.

Quantifying residue-specific conformational dynamics of a highly reactive 29-mer peptide.

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 kBT to 3.4 kBT), 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.