Integrated antibody and cellular immunity monitoring are required for assessment of the long term protection that will be essential for effective next generation vaccine development.

The COVID pandemic exposed the critical role T cells play in initial immunity, the establishment and maintenance of long term protection, and of durable responsiveness against novel viral variants. A growing body of evidence indicates that adding measures of cellular immunity will fill an important knowledge gap in vaccine clinical trials, likely leading to improvements in the effectiveness of the next generation vaccines against current and emerging variants. In depth cellular immune monitoring in Phase II trials, particularly for high risk populations such as the elderly or immune compromised, should result in better understanding of the dynamics and requirements for establishing effective long term protection. Such analyses can result in cellular immunity correlates that can then be deployed in Phase III studies using appropriate, scalable technologies. Measures of cellular immunity are less established than antibodies as correlates of clinical immunity, and some misconceptions persist about cellular immune monitoring usefulness, cost, complexity, feasibility, and scalability. We outline the currently available cellular immunity assays, review their readiness for use in clinical trials, their logistical requirements, and the type of information each assay generates. The objective is to provide a reliable source of information that could be leveraged to develop a rational approach for comprehensive immune monitoring during vaccine development.

Genes Involved in Maintaining Mitochondrial Membrane Potential Upon Electron Transport Chain Disruption.

Mitochondria are biosynthetic, bioenergetic, and signaling organelles with a critical role in cellular physiology. Dysfunctional mitochondria are associated with aging and underlie the cause of a wide range of diseases, from neurodegeneration to cancer. Through signaling, mitochondria regulate diverse biological outcomes. The maintenance of the mitochondrial membrane potential, for instance, is essential for proliferation, the release of mitochondrial reactive oxygen species, and oxygen sensing. The loss of mitochondrial membrane potential triggers pathways to clear damaged mitochondria and often results in cell death. In this study, we conducted a genome-wide positive selection CRISPR screen using a combination of mitochondrial inhibitors to uncover genes involved in sustaining a mitochondrial membrane potential, and therefore avoid cell death when the electron transport chain is impaired. Our screen identified genes involved in mitochondrial protein translation and ATP synthesis as essential for the induction of cell death when cells lose their mitochondrial membrane potential. This report intends to provide potential targets for the treatment of diseases associated with mitochondrial dysfunction.

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows 'hit' compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that 'hit' compounds are not artifactual.

Identification of Small-Molecule Inhibitors of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels function in the brain to limit neuronal excitability. Limiting the activity of these channels has been proposed as a therapy for major depressive disorder, but the critical role of HCN channels in cardiac pacemaking has limited efforts to develop therapies directed at the channel. Previous studies indicated that the function of HCN is tightly regulated by its auxiliary subunit, tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), which is not expressed in the heart. To target the function of the HCN channel in the brain without affecting the channel's function in the heart, we propose disrupting the interaction between HCN and TRIP8b. We developed a high-throughput fluorescence polarization (FP) assay to identify small molecules capable of disrupting this interaction. We used this FP assay to screen a 20,000-compound library and identified a number of active compounds. The active compounds were validated using an orthogonal AlphaScreen assay to identify one compound (0.005%) as the first confirmed hit for inhibiting the HCN-TRIP8b interaction. Identifying small molecules capable of disrupting the interaction between HCN and TRIP8b should enable the development of new research tools and small-molecule therapies that could benefit patients with depression.