mRNA vaccine platforms to prevent bacterial infections.

Bacterial infections are an urgent public health priority. The application of mRNA vaccine technology to prevent bacterial infections is a promising therapeutic strategy undergoing active development. This article discusses recent advances and limitations of mRNA vaccines to prevent bacterial diseases and provides perspectives on future research directions.

Corrigendum: Spatiotemporal analysis of 3D human iPSC-derived neural networks using a 3D multi-electrode array.

[This corrects the article DOI: 10.3389/fncel.2023.1287089.].

Spatiotemporal analysis of 3D human iPSC-derived neural networks using a 3D multi-electrode array.

While there is a growing appreciation of three-dimensional (3D) neural tissues (i.e., hydrogel-based, organoids, and spheroids), shown to improve cellular health and network activity to mirror brain-like activity in vivo, functional assessment using current electrophysiology techniques (e.g., planar multi-electrode arrays or patch clamp) has been technically challenging and limited to surface measurements at the bottom or top of the 3D tissue. As next-generation MEAs, specifically 3D MEAs, are being developed to increase the spatial precision across all three dimensions (X, Y, Z), development of improved computational analytical tools to discern region-specific changes within the Z dimension of the 3D tissue is needed. In the present study, we introduce a novel computational analytical pipeline to analyze 3D neural network activity recorded from a "bottom-up" 3D MEA integrated with a 3D hydrogel-based tissue containing human iPSC-derived neurons and primary astrocytes. Over a period of ~6.5 weeks, we describe the development and maturation of 3D neural activity (i.e., features of spiking and bursting activity) within cross sections of the 3D tissue, based on the vertical position of the electrode on the 3D MEA probe, in addition to network activity (identified using synchrony analysis) within and between cross sections. Then, using the sequential addition of postsynaptic receptor antagonists, bicuculline (BIC), 2-amino-5-phosphonovaleric acid (AP-5), and 6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), we demonstrate that networks within and between cross sections of the 3D hydrogel-based tissue show a preference for GABA and/or glutamate synaptic transmission, suggesting differences in the network composition throughout the neural tissue. The ability to monitor the functional dynamics of the entire 3D reconstructed neural tissue is a critical bottleneck; here we demonstrate a computational pipeline that can be implemented in studies to better interpret network activity within an engineered 3D neural tissue and have a better understanding of the modeled organ tissue.

Dose-dependent consequences of sub-chronic fentanyl exposure on neuron and glial co-cultures.

Fentanyl is one of the most common opioid analgesics administered to patients undergoing surgery or for chronic pain management. While the side effects of chronic fentanyl abuse are recognized (e.g., addiction, tolerance, impairment of cognitive functions, and inhibit nociception, arousal, and respiration), it remains poorly understood what and how changes in brain activity from chronic fentanyl use influences the respective behavioral outcome. Here, we examined the functional and molecular changes to cortical neural network activity following sub-chronic exposure to two fentanyl concentrations, a low (0.01 µM) and high (10 µM) dose. Primary rat co-cultures, containing cortical neurons, astrocytes, and oligodendrocyte precursor cells, were seeded in wells on either a 6-well multi-electrode array (MEA, for electrophysiology) or a 96-well tissue culture plate (for serial endpoint bulk RNA sequencing analysis). Once networks matured (at 28 days in vitro), co-cultures were treated with 0.01 or 10 µM of fentanyl for 4 days and monitored daily. Only high dose exposure to fentanyl resulted in a decline in features of spiking and bursting activity as early as 30 min post-exposure and sustained for 4 days in cultures. Transcriptomic analysis of the complex cultures after 4 days of fentanyl exposure revealed that both the low and high dose induced gene expression changes involved in synaptic transmission, inflammation, and organization of the extracellular matrix. Collectively, the findings of this in vitro study suggest that while neuroadaptive changes to neural network activity at a systems level was detected only at the high dose of fentanyl, transcriptomic changes were also detected at the low dose conditions, suggesting that fentanyl rapidly elicits changes in plasticity.

Cell-free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development.

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (<1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

A Survey of Preclinical Studies Evaluating Nanoparticle-Based Vaccines Against Non-Viral Sexually Transmitted Infections.

A worldwide estimate of over one million STIs are acquired daily and there is a desperate need for effective preventive as well as therapeutic measures to curtail this global health burden. Vaccines have been the most effective means for the control and potential eradication of infectious diseases; however, the development of vaccines against STIs has been a daunting task requiring extensive research for the development of safe and efficacious formulations. Nanoparticle-based vaccines represent a promising platform as they offer benefits such as targeted antigen presentation and delivery, co-localized antigen-adjuvant combinations for enhanced immunogenicity, and can be designed to be biologically inert. Here we discuss promising types of nanoparticles along with outcomes from nanoparticle-based vaccine preclinical studies against non-viral STIs including chlamydia, syphilis, gonorrhea, and recommendations for future nanoparticle-based vaccines against STIs.

Cellular, molecular, and therapeutic characterization of pilocarpine-induced temporal lobe epilepsy.

Animal models have expanded our understanding of temporal lobe epilepsy (TLE). However, translating these to cell-specific druggable hypotheses is not explored. Herein, we conducted an integrative insilico-analysis of an available transcriptomics dataset obtained from animals with pilocarpine-induced-TLE. A set of 119 genes with subtle-to-moderate impact predicted most forms of epilepsy with ~ 97% accuracy and characteristically mapped to upregulated homeostatic and downregulated synaptic pathways. The deconvolution of cellular proportions revealed opposing changes in diverse cell types. The proportion of nonneuronal cells increased whereas that of interneurons, except for those expressing vasoactive intestinal peptide (Vip), decreased, and pyramidal neurons of the cornu-ammonis (CA) subfields showed the highest variation in proportion. A probabilistic Bayesian-network demonstrated an aberrant and oscillating physiological interaction between nonneuronal cells involved in the blood-brain-barrier and Vip interneurons in driving seizures, and their role was evaluated insilico using transcriptomic changes induced by valproic-acid, which showed opposing effects in the two cell-types. Additionally, we revealed novel epileptic and antiepileptic mechanisms and predicted drugs using causal inference, outperforming the present drug repurposing approaches. These well-powered findings not only expand the understanding of TLE and seizure oscillation, but also provide predictive biomarkers of epilepsy, cellular and causal micro-circuitry changes associated with it, and a drug-discovery method focusing on these events.

Probing function in 3D neuronal cultures: A survey of 3D multielectrode array advances.

Recent advances in microphysiological systems have made significant strides to include design features that reconstruct key elements found in the brain, and in parallel advance technologies to detect the activity of electrogenic cells that form neural networks. In particular, three-dimensional multielectrode arrays (3D MEAs) are being developed with increasing levels of spatial and temporal precision, difficult to achieve with current 2D MEAs, insertable MEA probes, and/or optical imaging of calcium dynamics. Thus, providing a means to monitor the flow of neural network activity within all three dimensions (X, Y, and Z) of the engineered tissue. In the last 6 years, 3D MEAs, using either bottom-up or top-down designs, have been developed to overcome the current technical challenges in monitoring the functionality of the in vitro systems. Herein, we will report on the design and application of novel 3D MEA prototypes for probing neural activity throughout the 3D neural tissue.

Induction of Protection in Mice against a Chlamydia muridarum Respiratory Challenge by a Vaccine Formulated with the Major Outer Membrane Protein in Nanolipoprotein Particles.

Chlamydia trachomatis is a sexually transmitted bacterium that infects over 130 million individuals worldwide annually. To implement a vaccine, we developed a cell-free co-translational system to express the Chlamydia muridarum major outer membrane protein (MOMP). This approach uses a nanolipoprotein particles (tNLP) made from ApoA1 protein, amphiphilic telodendrimer and lipids that self-assemble to form 10-25 nm discs. These tNLP provide a protein-encapsulated lipid support to solubilize and fold membrane proteins. The cell-free system co-translated MOMP and ApoA1 in the presence of telodendrimer mixed with lipids. The MOMP-tNLP complex was amenable to CpG and FSL-1 adjuvant addition. To investigate the ability of MOMP-tNLP+CpG+FSL-1 to induce protection against an intranasal (i.n.) C. muridarum challenge, female mice were vaccinated intramuscularly (i.m.) or i.n. and i.m. simultaneously 4 weeks apart. Following vaccination with MOMP-tNLP+CpG+FSL-1, mice mounted significant humoral and cell-mediated immune responses. Following the i.n. challenge, mice vaccinated with MOMP-tNLP+CpG+FSL-1 i.n. + i.m. group were protected as determined by the percentage change in body weight and by the number of C. muridarum inclusion forming units (IFU) recovered from the lungs. To our knowledge, this is the first time a MOMP-based vaccine formulated in tNLP has been shown to protect against C. muridarum.

Intranasal Nanoparticle Vaccination Elicits a Persistent, Polyfunctional CD4 T Cell Response in the Murine Lung Specific for a Highly Conserved Influenza Virus Antigen That Is Sufficient To Mediate Protection from Influenza Virus Challenge.

Lung-localized CD4 T cells play a critical role in the control of influenza virus infection and can provide broadly protective immunity. However, current influenza vaccination strategies primarily target influenza hemagglutinin (HA) and are administered peripherally to induce neutralizing antibodies. We have used an intranasal vaccination strategy targeting the highly conserved influenza nucleoprotein (NP) to elicit broadly protective lung-localized CD4 T cell responses. The vaccine platform consists of a self-assembling nanolipoprotein particle (NLP) linked to NP with an adjuvant. We have evaluated the functionality, in vivo localization, and persistence of the T cells elicited. Our study revealed that intranasal vaccination elicits a polyfunctional subset of lung-localized CD4 T cells that persist long term. A subset of these lung CD4 T cells localize to the airway, where they can act as early responders following encounter with cognate antigen. Polyfunctional CD4 T cells isolated from airway and lung tissue produce significantly more effector cytokines IFN-γ and TNF-α, as well as cytotoxic functionality. When adoptively transferred to naive recipients, CD4 T cells from NLP:NP-immunized lung were sufficient to mediate 100% survival from lethal challenge with H1N1 influenza virus. IMPORTANCE Exploiting new, more efficacious strategies to potentiate influenza virus-specific immune responses is important, particularly for at-risk populations. We have demonstrated the promise of direct intranasal protein vaccination to establish long-lived immunity in the lung with CD4 T cells that possess features and positioning in the lung that are associated with both immediate and long-term immunity, as well as demonstrating direct protective potential.

Functional and transcriptional characterization of complex neuronal co-cultures.

Brain-on-a-chip systems are designed to simulate brain activity using traditional in vitro cell culture on an engineered platform. It is a noninvasive tool to screen new drugs, evaluate toxicants, and elucidate disease mechanisms. However, successful recapitulation of brain function on these systems is dependent on the complexity of the cell culture. In this study, we increased cellular complexity of traditional (simple) neuronal cultures by co-culturing with astrocytes and oligodendrocyte precursor cells (complex culture). We evaluated and compared neuronal activity (e.g., network formation and maturation), cellular composition in long-term culture, and the transcriptome of the two cultures. Compared to simple cultures, neurons from complex co-cultures exhibited earlier synapse and network development and maturation, which was supported by localized synaptophysin expression, up-regulation of genes involved in mature neuronal processes, and synchronized neural network activity. Also, mature oligodendrocytes and reactive astrocytes were only detected in complex cultures upon transcriptomic analysis of age-matched cultures. Functionally, the GABA antagonist bicuculline had a greater influence on bursting activity in complex versus simple cultures. Collectively, the cellular complexity of brain-on-a-chip systems intrinsically develops cell type-specific phenotypes relevant to the brain while accelerating the maturation of neuronal networks, important features underdeveloped in traditional cultures.

Characterization of Bacillus anthracis Spore Proteins Using a Nanoscaffold Vaccine Platform.

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required for sustained titers, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the NLP scaffold protein were detected. In summary, the NLP platform enhances humoral and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.

Modeling the temporal network dynamics of neuronal cultures.

Neurons form complex networks that evolve over multiple time scales. In order to thoroughly characterize these networks, time dependencies must be explicitly modeled. Here, we present a statistical model that captures both the underlying structural and temporal dynamics of neuronal networks. Our model combines the class of Stochastic Block Models for community formation with Gaussian processes to model changes in the community structure as a smooth function of time. We validate our model on synthetic data and demonstrate its utility on three different studies using in vitro cultures of dissociated neurons.

HIV influences microtubule associated protein-2: potential marker of HIV-associated neurocognitive disorders.

OBJECTIVE: Postmortem brains of patients diagnosed with HIV-1-associated neurocognitive disorders (HAND) exhibit loss of dendrites. However, the mechanisms by which synapses are damaged are not fully understood. DESIGN: Dendrite length and remodeling occurs via microtubules, the dynamics of which are regulated by microtubule-binding proteins, including microtubule-associated protein 2 (MAP2). The HIV protein gp120 is neurotoxic and interferes with neuronal microtubules. We measured MAP2 concentrations in human cerebrospinal fluid (CSF) and MAP2 immunoreactivity in rat cortical neurons exposed to HIV and gp120. METHODS: First, we examined whether HIV affects MAP2 levels by analyzing the CSF of 27 persons living with HIV (PLH) whose neurocognitive performance had been characterized. We then used rat cortical neurons to study the mechanisms of HIV-mediated dendritic loss. RESULTS: PLH who had HAND had greater MAP2 concentrations within the CSF than cognitive normal PLH. In cortical neurons, the deleterious effect of HIV on MAP2-positive dendrites occurred through a gp120-mediated mechanism. The neurotoxic effect of HIV was blocked by a CCR5 antagonist and prevented by Helix-A, a peptide that displaces gp120 from binding to microtubules, conjugated to a nanolipoprotein particle delivery platform. CONCLUSION: Our findings support that HIV at least partially effects its neurotoxicity via neuronal cytoskeleton modifications and provide evidence of a new therapeutic compound that could be used to prevent the HIV-associated neuropathology.

A flexible 3-dimensional microelectrode array for in vitro brain models.

Three-dimensional (3D) in vitro models have become increasingly popular as systems to study cell-cell and cell-ECM interactions dependent on the spatial, mechanical, and chemical cues within the environment of the tissue, which is limited in traditional two-dimensional (2D) models. Although electrophysiological recordings of neuronal action potentials through 2D microelectrode arrays (MEAs) are a common and trusted method of evaluating neuronal function, network communication, and response to chemicals and biologicals, there are currently limited options for measuring electrophysiological activity from many locations simultaneously throughout a 3D network of neurons in vitro. Here, we have developed a thin-film, 3D flexible microelectrode array (3DMEA) that non-invasively interrogates a 3D culture of neurons and can accommodate 256 channels of recording or stimulation. Importantly, the 3DMEA is straightforward to fabricate and integrates with standard commercially available electrophysiology hardware. Polyimide probe arrays were microfabricated on glass substrates and mechanically actuated to collectively lift the arrays into a vertical position, relying solely on plastic deformation of their base hinge regions to maintain vertical alignment. Human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes were entrapped in a collagen-based hydrogel and seeded onto the 3DMEA, enabling growth of suspended cells in the matrix and the formation and maturation of a neural network around the 3DMEA probes. The 3DMEA supported the growth of functional neurons in 3D with action potential spike and burst activity recorded over 45 days in vitro. This platform is an important step in facilitating noninvasive electrophysiological characterization of 3D networks of electroactive cells in vitro.

Cationic HDL mimetics enhance in vivo delivery of self-replicating mRNA.

In vivo delivery of large RNA molecules has significant implications for novel gene therapy, biologics delivery, and vaccine applications. We have developed cationic nanolipoprotein particles (NLPs) to enhance the complexation and delivery of large self-amplifying mRNAs (replicons) in vivo. NLPs are high-density lipoprotein (HDL) mimetics, comprised of a discoidal lipid bilayer stabilized by apolipoproteins that are readily functionalized to provide a versatile delivery platform. Herein, we systematically screened NLP assembly with a wide range of lipidic and apolipoprotein constituents, using biophysical metrics to identify lead candidates for in vivo RNA delivery. NLPs formulated with cationic lipids successfully complexed with RNA replicons encoding luciferase, provided measurable protection from RNase degradation, and promoted replicon in vivo expression. The NLP complexation of the replicon and in vivo transfection efficiency were further enhanced by modulating the type and percentage of cationic lipid, the ratio of cationic NLP to replicon, and by incorporating additive molecules.

Optimizing cell encapsulation condition in ECM-Collagen I hydrogels to support 3D neuronal cultures.

BACKGROUND: The emergence of three-dimensional (3D) cell culture in neural tissue engineering has significantly elevated the complexity and relevance of in vitro systems. This is due in large part to the incorporation of biomaterials to impart structural dimensionality on the neuronal cultures. However, a comprehensive understanding of how key seeding parameters affect changes in cell distribution and viability remain unreported. NEW METHOD: In this study, we systematically evaluated permutations in seeding conditions (i.e., cell concentration and atmospheric CO2 levels) to understand how these affect key parameters in 3D culture characterization (i.e., cell health and distribution). Primary rat cortical neurons (i.e., 2 × 106, 4 × 106, and 1 × 107 cells/mL) were entrapped in collagen blended with ECM proteins (ECM-Collagen) and exposed to atmospheric CO2 (i.e., 0 vs 5% CO2) during fibrillogenesis. RESULTS: At 14 days in vitro (DIV), cell distribution within the hydrogel was dependent on cell concentration and atmospheric CO2 during fibrillogenesis. A uniform distribution of cells was observed in cultures with 2 × 106 and 4 × 106 cells/mL in the presence of 5% CO2, while a heterogeneous distribution was observed in cultures with 1 × 107 cells/mL or in the absence of CO2. Furthermore, increased cell concentration was proportional to the rise in cell death at 14 DIV, although cells remain viable >30 DIV. COMPARISON WITH EXISTING METHODS: ECM-Collagen gels have been shown to increase cell viability of neurons long-term. CONCLUSION: In using ECM-collagen gels, we highlight the importance of optimizing seeding parameters and thorough 3D culture characterization to understand the neurophysiological responses of these 3D systems.

A Reconfigurable In Vitro Model for Studying the Blood-Brain Barrier.

Much of what is currently known about the role of the blood-brain barrier (BBB) in regulating the passage of chemicals from the blood stream to the central nervous system (CNS) comes from animal in vivo models (requiring extrapolation to human relevance) and 2D static in vitro systems, which fail to capture the rich cell-cell and cell-matrix interactions of the dynamic 3D in vivo tissue microenvironment. In this work we have developed a BBB platform that allows for a high degree of customization in cellular composition, cellular orientation, and physiologically-relevant fluid dynamics. The system characterized and presented in this study reproduces key characteristics of a BBB model (e.g. tight junctions, efflux pumps) allowing for the formation of a selective and functional barrier. We demonstrate that our in vitro BBB is responsive to both biochemical and mechanical cues. This model further allows for culture of a CNS-like space around the BBB. The design of this platform is a valuable tool for studying BBB function as well as for screening of novel therapeutics.

Tissue-specific extracellular matrix accelerates the formation of neural networks and communities in a neuron-glia co-culture on a multi-electrode array.

The brain's extracellular matrix (ECM) is a macromolecular network composed of glycosaminoglycans, proteoglycans, glycoproteins, and fibrous proteins. In vitro studies often use purified ECM proteins for cell culture coatings, however these may not represent the molecular complexity and heterogeneity of the brain's ECM. To address this, we compared neural network activity (over 30 days in vitro) from primary neurons co-cultured with glia grown on ECM coatings from decellularized brain tissue (bECM) or MaxGel, a non-tissue-specific ECM. Cells were grown on a multi-electrode array (MEA) to enable noninvasive long-term interrogation of neuronal networks. In general, the presence of ECM accelerated the formation of networks without affecting the inherent network properties. However, specific features of network activity were dependent on the type of ECM: bECM enhanced network activity over a greater region of the MEA whereas MaxGel increased network burst rate associated with robust synaptophysin expression. These differences in network activity were not attributable to cellular composition, glial proliferation, or astrocyte phenotypes, which remained constant across experimental conditions. Collectively, the addition of ECM to neuronal cultures represents a reliable method to accelerate the development of mature neuronal networks, providing a means to enhance throughput for routine evaluation of neurotoxins and novel therapeutics.

Extent of MHC Clustering Regulates Selectivity and Effectiveness of T Cell Responses.

MHC proteins that present peptide ligands for recognition by TCR form nanoscale clusters on the cell membrane of APCs. How the extent of MHC clustering controls productive TCR engagement and TCR-mediated signaling has not been systematically studied. To evaluate the role of MHC clustering, we exploited nanoscale discoidal membrane mimetics (nanolipoprotein particles) to capture and present peptide-MHC (pMHC) ligands at various densities. We examined the binding of these model membrane clusters to the surface of live human CD8+ T cells and the subsequent triggering of intracellular signaling. The data demonstrate that the proximity of pMHC ligands, high association rate of CD8-MHC interactions, and relatively long lifetime of cognate TCR-pMHC complexes emerge as essential parameters, explaining the significance of MHC clustering. Rapid rebinding of CD8 to MHC suggests a dual role of CD8 in facilitating the T cells' hunt for a rare foreign pMHC ligand and the induction of rapid T cell response. Thus, our findings provide a new understanding of how MHC clustering influences multivalent interactions of pMHC ligands with CD8 and TCR on live T cells that regulate Ag recognition, kinetics of intracellular signaling, and the selectivity and efficiency of T cell responses.