Protocol for localized macrophage stimulation with small-molecule TLR agonist via fluidic force microscopy.

Here, we present a protocol to deliver nanoliter volumes of Toll-like receptor (TLR) agonist onto a culture of nuclear factor κB (NF-κB) reporter macrophages using fluidic force microscopy and a micron-scale probe. We describe steps for quantifying the dose of agonist by modeling their diffusion with experimental inputs. We then detail procedures for quantifying and categorizing macrophage responses to individual and varied doses and combining agonist concentration and macrophage response to analyze the NF-κB response to localized TLR stimulation. For complete details on the use and execution of this protocol, please refer to Mulder et al. (2024).1.

Immune Potentiation of PLGA Controlled-Release Vaccines for Improved Immunological Outcomes.

With the emergence of SARS-CoV-2 and the continued emergence of new infectious diseases, there is a need to improve and expand current vaccine technology. Controlled-release subunit vaccines provide several benefits over current vaccines on the market, including the use of less antigen and fewer boost doses. Previously, our group reported molecules that alter NF-κB signaling improved the vaccine's performance and improved adjuvant-related tolerability. In this report, we test how these immune potentiators will influence responses when included as part of a controlled-release poly(lactic-co-glycolic) vaccine formulation. Murine in vivo studies revealed that SN50 and honokiol improved antibody levels at early vaccine time points. Microparticles with SN50 produced strong antibody levels over a longer period compared to microparticles without SN50. The same particles also increased T-cell activity. All of the immune potentiators tested further promoted Th2 humoral responses already exhibited by the control CpG OVA microparticle formulation. Overall, under controlled-release conditions, immune potentiators enhance the existing effects of controlled-release formulations, making it a potentially beneficial additive for controlled-release vaccine formulations.

Evidence of collective influence in innate sensing using fluidic force microscopy.

The innate immune system initiates early response to infection by sensing molecular patterns of infection through pattern-recognition receptors (PRRs). Previous work on PRR stimulation of macrophages revealed significant heterogeneity in single cell responses, suggesting the importance of individual macrophage stimulation. Current methods either isolate individual macrophages or stimulate a whole culture and measure individual readouts. We probed single cell NF-κB responses to localized stimuli within a naïve culture with Fluidic Force Microscopy (FluidFM). Individual cells stimulated in naïve culture were more sensitive compared to individual cells in uniformly stimulated cultures. In cluster stimulation, NF-κB activation decreased with increased cell density or decreased stimulation time. Our results support the growing body of evidence for cell-to-cell communication in macrophage activation, and limit potential mechanisms. Such a mechanism might be manipulated to tune macrophage sensitivity, and the density-dependent modulation of sensitivity to PRR signals could have relevance to biological situations where macrophage density increases.

Polymer Patterning by Laser-Induced Multipoint Initiation of Frontal Polymerization.

Frontal polymerization (FP) is an approach for thermosetting plastics at a lower energy cost than an autoclave. The potential to generate simultaneous propagation of multiple polymerization fronts has been discussed as an exciting possibility. However, FP initiated at more than two points simultaneously has not been demonstrated. Multipoint initiation could enable both large-scale material fabrication and unique pattern generation. Here, the authors present laser-patterned photothermal heating as a method for simultaneous initiation of FP at multiple locations in a 2-D sample. Carbon black particles are mixed into liquid resin (dicyclopentadiene) to enhance absorption of light from a Ti:sapphire laser (800 nm) focused on a sample. The laser is time-shared by rapid steering among initiation points, generating polymerization using up to seven simultaneous points of initiation. This process results in the formation of both symmetric and asymmetric seam patterns resulting from the collision of fronts. The authors also present and validate a theoretical framework for predicting the seam patterns formed by front collisions. This framework allows the design of novel patterns via an inverse solution for determining the initiation points required to form a desired pattern. Future applications of this approach could enable rapid, energy-efficient manufacturing of novel composite-like patterned materials.

Bioinspired mechanical mineralization of organogels.

Mineralization is a long-lasting method commonly used by biological materials to selectively strengthen in response to site specific mechanical stress. Achieving a similar form of toughening in synthetic polymer composites remains challenging. In previous work, we developed methods to promote chemical reactions via the piezoelectrochemical effect with mechanical responses of inorganic, ZnO nanoparticles. Herein, we report a distinct example of a mechanically-mediated reaction in which the spherical ZnO nanoparticles react themselves leading to the formation of microrods composed of a Zn/S mineral inside an organogel. The microrods can be used to selectively create mineral deposits within the material resulting in the strengthening of the overall resulting composite.

Stress-activated friction in sheared suspensions probed with piezoelectric nanoparticles.

A hallmark of concentrated suspensions is non-Newtonian behavior, whereby the viscosity increases dramatically once a characteristic shear rate or stress is exceeded. Such strong shear thickening is thought to originate from a network of frictional particle-particle contact forces, which forms under sufficiently large stress, evolves dynamically, and adapts to changing loads. While there is much evidence from simulations for the emergence of this network during shear thickening, experimental confirmation has been difficult. Here, we use suspensions of piezoelectric nanoparticles and exploit the strong local stress focusing within the network to activate charge generation. This charging can then be detected in the measured ac conductance and serve as a signature of frictional contact formation. The direct link between stress-activated frictional particle interactions and piezoelectric suspension response is further demonstrated by tracking the emergence of structural memory in the contact network under oscillatory shear and by showing how stress-activated friction can drive mechano-transduction of chemical reactions with nonlinear reaction kinetics. Taken together, this makes the ac conductance of piezoelectric suspensions a sensitive in-situ reporter of the micromechanics associated with frictional interactions.

Data-driven discovery of innate immunomodulators via machine learning-guided high throughput screening.

The innate immune response is vital for the success of prophylactic vaccines and immunotherapies. Control of signaling in innate immune pathways can improve prophylactic vaccines by inhibiting unfavorable systemic inflammation and immunotherapies by enhancing immune stimulation. In this work, we developed a machine learning-enabled active learning pipeline to guide in vitro experimental screening and discovery of small molecule immunomodulators that improve immune responses by altering the signaling activity of innate immune responses stimulated by traditional pattern recognition receptor agonists. Molecules were tested by in vitro high throughput screening (HTS) where we measured modulation of the nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB) and the interferon regulatory factors (IRF) pathways. These data were used to train data-driven predictive models linking molecular structure to modulation of the NF-κB and IRF responses using deep representational learning, Gaussian process regression, and Bayesian optimization. By interleaving successive rounds of model training and in vitro HTS, we performed an active learning-guided traversal of a 139 998 molecule library. After sampling only ∼2% of the library, we discovered viable molecules with unprecedented immunomodulatory capacity, including those capable of suppressing NF-κB activity by up to 15-fold, elevating NF-κB activity by up to 5-fold, and elevating IRF activity by up to 6-fold. We extracted chemical design rules identifying particular chemical fragments as principal drivers of specific immunomodulation behaviors. We validated the immunomodulatory effect of a subset of our top candidates by measuring cytokine release profiles. Of these, one molecule induced a 3-fold enhancement in IFN-ß production when delivered with a cyclic di-nucleotide stimulator of interferon genes (STING) agonist. In sum, our machine learning-enabled screening approach presents an efficient immunomodulator discovery pipeline that has furnished a library of novel small molecules with a strong capacity to enhance or suppress innate immune signaling pathways to shape and improve prophylactic vaccination and immunotherapies.

Understanding How Cationic Polymers' Properties Inform Toxic or Immunogenic Responses via Parametric Analysis.

Cationic polymers are widely used materials in diverse biotechnologies. Subtle variations in these polymers' properties can change them from exceptional delivery agents to toxic inflammatory hazards. Conventional screening strategies optimize for function in a specific application rather than observing how underlying polymer-cell interactions emerge from polymers' properties. An alternative approach is to map basic underlying responses, such as immunogenicity or toxicity, as a function of basic physicochemical parameters to inform the design of materials for a breadth of applications. To demonstrate the potential of this approach, we synthesized 107 polymers varied in charge, hydrophobicity, and molecular weight. We then screened this library for cytotoxic behavior and immunogenic responses to map how these physicochemical properties inform polymer-cell interactions. We identify three compositional regions of interest and use confocal microscopy to uncover the mechanisms behind the observed responses. Finally, immunogenic activity is confirmed in vivo. Highly cationic polymers disrupted the cellular plasma membrane to induce a toxic phenotype, while high molecular weight, hydrophobic polymers were uptaken by active transport to induce NLRP3 inflammasome activation, an immunogenic phenotype. Tertiary amine- and triethylene glycol-containing polymers did not invoke immunogenic or toxic responses. The framework described herein allows for the systematic characterization of new cationic materials with different physicochemical properties for applications ranging from drug and gene delivery to antimicrobial coatings and tissue scaffolds.

Lung cDC1 and cDC2 dendritic cells priming naive CD8+ T cells in situ prior to migration to draining lymph nodes.

The current paradigm indicates that naive T cells are primed in secondary lymphoid organs. Here, we present evidence that intranasal administration of peptide antigens appended to nanofibers primes naive CD8+ T cells in the lung independently and prior to priming in the draining mediastinal lymph node (MLN). Notably, comparable accumulation and transcriptomic responses of CD8+ T cells in lung and MLN are observed in both Batf3KO and wild-type (WT) mice, indicating that, while cDC1 dendritic cells (DCs) are the major subset for cross-presentation, cDC2 DCs alone are capable of cross-priming CD8+ T cells both in the lung and draining MLN. Transcription analyses reveal distinct transcriptional responses in lung cDC1 and cDC2 to intranasal nanofiber immunization. However, both DC subsets acquire shared transcriptional responses upon migration into the lymph node, thus uncovering a stepwise activation process of cDC1 and cDC2 toward their ability to cross-prime effector and functional memory CD8+ T cell responses.

Cell-targeted vaccines: implications for adaptive immunity.

Recent advancements in immunology and chemistry have facilitated advancements in targeted vaccine technology. Targeting specific cell types, tissue locations, or receptors can allow for modulation of the adaptive immune response to vaccines. This review provides an overview of cellular targets of vaccines, suggests methods of targeting and downstream effects on immune responses, and summarizes general trends in the literature. Understanding the relationships between vaccine targets and subsequent adaptive immune responses is critical for effective vaccine design. This knowledge could facilitate design of more effective, disease-specialized vaccines.

Next-Generation Adjuvants: Applying Engineering Methods to Create and Evaluate Novel Immunological Responses.

Adjuvants are a critical component of vaccines. Adjuvants typically target receptors that activate innate immune signaling pathways. Historically, adjuvant development has been laborious and slow, but has begun to accelerate over the past decade. Current adjuvant development consists of screening for an activating molecule, formulating lead molecules with an antigen, and testing this combination in an animal model. There are very few adjuvants approved for use in vaccines, however, as new candidates often fail due to poor clinical efficacy, intolerable side effects, or formulation limitations. Here, we consider new approaches using tools from engineering to improve next-generation adjuvant discovery and development. These approaches will create new immunological outcomes that will be evaluated with novel diagnostic tools. Potential improved immunological outcomes include reduced vaccine reactogenicity, tunable adaptive responses, and enhanced adjuvant delivery. Evaluations of these outcomes can leverage computational approaches to interpret "big data" obtained from experimentation. Applying engineering concepts and solutions will provide alternative perspectives, further accelerating the field of adjuvant discovery.

Nanovaccine that activates the NLRP3 inflammasome enhances tumor specific activation of anti-cancer immunity.

Neoantigen cancer vaccines that target tumor specific mutations are emerging as a promising modality for cancer immunotherapy. To date, various approaches have been adopted to enhance efficacy of these therapies, but the low immunogenicity of neoantigens has hindered clinical application. To address this challenge, we developed a polymeric nanovaccine platform that activates the NLRP3 inflammasome, a key immunological signaling pathway in pathogen recognition and clearance. The nanovaccine is comprised of a poly (orthoester) scaffold engrafted with a small-molecule TLR7/8 agonist and an endosomal escape peptide that facilitates lysosomal rupture and NLRP3 inflammasome activation. Upon solvent transfer, the polymer self-assembles with neoantigens to form ∼50 nm nanoparticles that facilitate co-delivery to antigen-presenting cells. This polymeric activator of the inflammasome (PAI) was found to induce potent antigen-specific CD8+ T cell responses characterized by IFN-γ and GranzymeB secretion. Moreover, in combination with immune checkpoint blockade therapy, the nanovaccine stimulated robust anti-tumor immune responses against established tumors in EG.7-OVA, B16·F10, and CT-26 models. Results from our studies indicate that NLRP3 inflammasome activating nanovaccines demonstrate promise for development as a robust platform to enhance immunogenicity of neoantigen therapies.

Discovery of New States of Immunomodulation for Vaccine Adjuvants via High Throughput Screening: Expanding Innate Responses to PRRs.

Stimulation of the innate immune system is crucial in both effective vaccinations and immunotherapies. This is often achieved through adjuvants, molecules that usually activate pattern recognition receptors (PRRs) and stimulate two innate immune signaling pathways: the nuclear factor kappa-light-chain-enhancer of activated B-cells pathway (NF-κB) and the interferon regulatory factors pathway (IRF). Here, we demonstrate the ability to alter and improve adjuvant activity via the addition of small molecule "immunomodulators". By modulating signaling activity instead of receptor binding, these molecules allow the customization of select innate responses. We demonstrate both inhibition and enhancement of the products of the NF-κB and IRF pathways by several orders of magnitude. Some modulators apply generally across many receptors, while others focus specifically on individual receptors. Modulators boost correlates of a protective immune responses in a commercial flu vaccine model and reduced correlates of reactogenicity in a typhoid vaccine model. These modulators have a range of applications: from adjuvanticity in prophylactics to enhancement of immunotherapy.

Size matters in non-canonical inflammasome activation and cell-mediated immunity.

Particulate adjuvants are key components of many approved vaccines, but their mechanism of adjuvanticity is debated. Muñoz-Wolf et al.1 find that 50-nm particles maximize cell-mediated immune responses by activating the caspase-11 inflammasome, providing mechanistic insight to particulate adjuvant technologies.

Immunostimulatory Polymers as Adjuvants, Immunotherapies, and Delivery Systems.

Activating innate immunity in a controlled manner is necessary for the development of next-generation therapeutics. Adjuvants, or molecules that modulate the immune response, are critical components of vaccines and immunotherapies. While small molecules and biologics dominate the adjuvant market, emerging evidence supports the use of immunostimulatory polymers in therapeutics. Such polymers can stabilize and deliver cargo while stimulating the immune system by functioning as pattern recognition receptor (PRR) agonists. At the same time, in designing polymers that engage the immune system, it is important to consider any unintended initiation of an immune response that results in adverse immune-related events. Here, we highlight biologically derived and synthetic polymer scaffolds, as well as polymer-adjuvant systems and stimuli-responsive polymers loaded with adjuvants, that can invoke an immune response. We present synthetic considerations for the design of such immunostimulatory polymers, outline methods to target their delivery, and discuss their application in therapeutics. Finally, we conclude with our opinions on the design of next-generation immunostimulatory polymers, new applications of immunostimulatory polymers, and the development of improved preclinical immunocompatibility tests for new polymers.

Temporal Control of Trained Immunity via Encapsulated Release of ß-Glucan Improves Therapeutic Applications.

Emerging diseases require generating new vaccines, which can often be time consuming. An alternate method to boost host defense is by inducing nonspecific innate immune memory, called trained immunity, to develop novel prophylactics. Many molecules, most notably ß-glucan, induce trained immunity, but their effects are often short-lived and uncontrolled. This lack of temporal control limits both the therapeutic ability of training and provides fundamental questions about its nature. To achieve temporal control of trained immunity, controlled release nanoparticles encapsulating only 3.5% of the standard dose of ß-glucan to attain sustained release over a month are engineered. Nanoparticle-trained mice exhibit prolonged training effects and improve resistance to a B16F10 tumor challenge compared to mice that receive an equivalent amount of free ß-glucan. The duration of trained immunity is further fine tuned by synthesizing nanoparticles composed of different molecular weights to modulate the release kinetics. These results demonstrate that dosing and temporal control can substantially alter the trained response to unanticipated levels. As such, this approach using sustained release platforms might lead to a novel prophylactic strategy for improved disease resistance against a wide variety of diseases.

Improving the Adjuvanticity of Small Molecule Immune Potentiators Using Covalently Linked NF-κB Modulators.

Small molecule immune potentiators (SMIPs) such as imidazoquinolinone derivatives that activate Toll-like receptor (TLR) 7/8 have immense potential as vaccine adjuvants and as antitumor agents. However, these molecules have high bioavailability that results in unacceptable levels of systemic inflammation due to adjuvant toxicity, thereby greatly limiting their use. To address this challenge, here we report the design and synthesis of novel imidazoquinolinone-NF-κB immunomodulator dimers. Employing in vitro assays, we screened a select library of synthesized dimers and selected viable candidates for further in vivo experiments. With ovalbumin as a model antigen, we vaccinated mice and demonstrated that these dimers reduce the systemic toxicity associated with SMIPs to baseline levels while simultaneously maintaining the adjuvanticity in a vaccine formulation. Additionally, we showed that select dimers improved efficacy in a CT26 mouse colon carcinoma tumor model while eliciting minimal adjuvant toxicity.

Controllable Frontal Polymerization and Spontaneous Patterning Enabled by Phase-Changing Particles.

Frontal polymerization provides a rapid, economic, and environmentally friendly methodology to manufacture thermoset polymers and composites. Despite its efficiency and reduced environmental impact, the manufacturing method is underutilized due to the limited fundamental understanding of its dynamic control. This work reports the control and patterning of the front propagation in a dicyclopentadiene resin by immersion of phase-changing polycaprolactone particles. Predictive and designed patterning is enabled by multiphysical numerical analyses, which reveal that the interplay between endothermic phase transition, exothermic chemical reaction, and heat exchange govern the temperature, velocity, and propagation path of the front via two different interaction regimes. To pattern the front, one can vary the size and spacing between the particles and increase the number of propagating fronts, resulting in tunable physical patterns formed due to front separation and merging near the particles. Both single- and double-frontal polymerization experiments in an open mold are performed. The results confirm the front-particle interaction mechanisms and the shapes of the patterns explored numerically. The present study offers a fundamental understanding of frontal polymerization in the presence of heat-absorbing second-phase materials and proposes a potential one-step manufacturing method for precisely patterned polymeric and composite materials without masks, molds, or printers.

Manipulating Frontal Polymerization and Instabilities with Phase-Changing Microparticles.

Recently presented as a rapid and eco-friendly manufacturing method for thermoset polymers and composites, frontal polymerization (FP) experiences thermo-chemical instabilities under certain conditions, leading to visible patterns and spatially dependent material properties. Through numerical analyses and experiments, we demonstrate how the front velocity, temperature, and instability in the frontal polymerization of cyclooctadiene are affected by the presence of poly(caprolactone) microparticles homogeneously mixed with the resin. The phase transformation associated with the melting of the microparticles absorbs some of the exothermic reaction energy generated by the FP, reduces the amplitude and order of the thermal instabilities, and suppresses the front velocity and temperatures. Experimental measurements validate predictions of the dependence of the front velocity and temperature on the microparticle volume fraction provided by the proposed homogenized reaction-diffusion model.

A synthetic pathogen mimetic molecule induces a highly amplified synergistic immune response via activation of multiple signaling pathways.

The current understanding of how the immune system processes complex information during natural infections is yet to be exploited for the molecular design of potent immune activators. Here, we address this challenge by design of a pathogen-mimetic molecule that simultaneously co-activates cell-surface active, endosomal and cytosolic immune receptors.