Integrating Single Domain Antibodies into Field-Deployable Rapid Assays.

Single domain antibodies (sdAb) are the recombinant variable heavy domains derived from camelid heavy-chain antibodies. While they have binding affinities equivalent to conventional antibodies, sdAb are only one-tenth the size and possess numerous advantages such as excellent thermal stability with the ability to refold following denaturation, and inexpensive production in Escherichia coli or yeast. However, their small size does have drawbacks, one being that they can lose activity upon attachment or adsorption to surfaces, or may fail to adsorb efficiently, as they are highly soluble. This can make the transition from using conventional antibodies to sdAb nontrivial for assay development. Specifically, it is often necessary to re-optimize the protocols and tailor the recombinant sdAb through protein engineering to function efficiently in handheld assays, which currently are utilized for point of care testing and field applications. This work focuses on optimizing the integration of sdAb into rapid vertical flow assays. To achieve this goal, we engineered sdAb-based constructs and developed general protocols for the attachment of the sdAb to both gold nanoparticles and a support membrane. We achieved a limit of detection of 0.11 µg/mL for toxins staphylococcal enterotoxin B and ricin, both potential biothreat agents. Additionally, we demonstrated the ability to detect the nucleocapsid protein of SARS-CoV-2, a common target of antigen tests for COVID-19.

Single-Domain Antibodies for the Detection of SARS-CoV-2 Nucleocapsid Protein.

The goal of this work was to develop recombinantly expressed variable domains derived from camelid heavy-chain antibodies known as single-domain antibodies (sdAbs) directed against the SARS-CoV-2 nucleocapsid protein for incorporation into detection assays. To achieve this, a llama was immunized using a recombinant SARS-CoV-2 nucleocapsid protein and an immune phage-display library of variable domains was developed. The sdAbs selected from this library segregated into five distinct sequence families. Three of these families bind to unique epitopes with high affinity, low nM to sub-nM KD, as determined by surface plasmon resonance. To further enhance the utility of these sdAbs for the detection of nucleocapsid protein, homobivalent and heterobivalent genetic fusion constructs of the three high-affinity sdAbs were prepared. The effectiveness of the sdAbs for the detection of nucleocapsid protein was evaluated using MagPlex fluid array assays, a multiplexed immunoassay on color-coded magnetic microspheres. Using the optimal bivalent pair, one immobilized on the microsphere and the other serving as the biotinylated recognition reagent, a detection limit as low as 50 pg/mL of recombinant nucleocapsid and of killed virus down to 1.28 × 103 pfu/mL was achieved. The sdAbs described here represent immune reagents that can be tailored to be optimized for a number of detection platforms and may one day aid in the detection of SARS-CoV-2 to assist in controlling the current pandemic.

Tryptophan Metabolites and Aryl Hydrocarbon Receptor in Severe Acute Respiratory Syndrome, Coronavirus-2 (SARS-CoV-2) Pathophysiology.

The metabolism of tryptophan is intimately associated with the differential regulation of diverse physiological processes, including in the regulation of responses to severe acute respiratory syndrome, coronavirus-2 (SARS-CoV-2) infection that underpins the COVID-19 pandemic. Two important products of tryptophan metabolism, viz kynurenine and interleukin (IL)4-inducible1 (IL41)-driven indole 3 pyruvate (I3P), activate the aryl hydrocarbon receptor (AhR), thereby altering the nature of immune responses to SARS-CoV-2 infection. AhR activation dysregulates the initial pro-inflammatory cytokines production driven by neutrophils, macrophages, and mast cells, whilst AhR activation suppresses the endogenous antiviral responses of natural killer cells and CD8+ T cells. Such immune responses become further dysregulated by the increased and prolonged pro-inflammatory cytokine suppression of pineal melatonin production coupled to increased gut dysbiosis and gut permeability. The suppression of pineal melatonin and gut microbiome-derived butyrate, coupled to an increase in circulating lipopolysaccharide (LPS) further dysregulates the immune response. The AhR mediates its effects via alterations in the regulation of mitochondrial function in immune cells. The increased risk of severe/fatal SARS-CoV-2 infection by high risk conditions, such as elderly age, obesity, and diabetes are mediated by these conditions having expression levels of melatonin, AhR, butyrate, and LPS that are closer to those driven by SARS-CoV-2 infection. This has a number of future research and treatment implications, including the utilization of melatonin and nutraceuticals that inhibit the AhR, including the polyphenols, epigallocatechin gallate (EGCG), and resveratrol.

Tumour Microenvironment: Roles of the Aryl Hydrocarbon Receptor, O-GlcNAcylation, Acetyl-CoA and Melatonergic Pathway in Regulating Dynamic Metabolic Interactions across Cell Types-Tumour Microenvironment and Metabolism.

This article reviews the dynamic interactions of the tumour microenvironment, highlighting the roles of acetyl-CoA and melatonergic pathway regulation in determining the interactions between oxidative phosphorylation (OXPHOS) and glycolysis across the array of cells forming the tumour microenvironment. Many of the factors associated with tumour progression and immune resistance, such as yin yang (YY)1 and glycogen synthase kinase (GSK)3ß, regulate acetyl-CoA and the melatonergic pathway, thereby having significant impacts on the dynamic interactions of the different types of cells present in the tumour microenvironment. The association of the aryl hydrocarbon receptor (AhR) with immune suppression in the tumour microenvironment may be mediated by the AhR-induced cytochrome P450 (CYP)1b1-driven 'backward' conversion of melatonin to its immediate precursor N-acetylserotonin (NAS). NAS within tumours and released from tumour microenvironment cells activates the brain-derived neurotrophic factor (BDNF) receptor, TrkB, thereby increasing the survival and proliferation of cancer stem-like cells. Acetyl-CoA is a crucial co-substrate for initiation of the melatonergic pathway, as well as co-ordinating the interactions of OXPHOS and glycolysis in all cells of the tumour microenvironment. This provides a model of the tumour microenvironment that emphasises the roles of acetyl-CoA and the melatonergic pathway in shaping the dynamic intercellular metabolic interactions of the various cells within the tumour microenvironment. The potentiation of YY1 and GSK3ß by O-GlcNAcylation will drive changes in metabolism in tumours and tumour microenvironment cells in association with their regulation of the melatonergic pathway. The emphasis on metabolic interactions across cell types in the tumour microenvironment provides novel future research and treatment directions.

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme.

There are currently few approved effective treatments for SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Nanobodies are 12-15 kDa single-domain antibody fragments that can be delivered by inhalation and are amenable to relatively inexpensive large scale production compared to other biologicals. We have isolated nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1-5 nM affinity. The lead nanobody candidate, NIH-CoVnb-112, blocks SARS-CoV-2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC50 of 0.3 µg/mL. NIH-CoVnb-112 retains structural integrity and potency after nebulization. Furthermore, NIH-CoVnb-112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein. These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.

Aryl Hydrocarbon Receptor Role in Co-Ordinating SARS-CoV-2 Entry and Symptomatology: Linking Cytotoxicity Changes in COVID-19 and Cancers; Modulation by Racial Discrimination Stress.

There is an under-recognized role of the aryl hydrocarbon receptor (AhR) in co-ordinating the entry and pathophysiology of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) that underpins the COVID-19 pandemic. The rise in pro-inflammatory cytokines during the 'cytokine storm' induce indoleamine 2,3-dioxygenase (IDO), leading to an increase in kynurenine that activates the AhR, thereby heightening the initial pro-inflammatory cytokine phase and suppressing the endogenous anti-viral response. Such AhR-driven changes underpin the heightened severity and fatality associated with pre-existent high-risk medical conditions, such as type II diabetes, as well as to how racial discrimination stress contributes to the raised severity/fatality in people from the Black Asian and Minority Ethnic (BAME) communities. The AhR is pivotal in modulating mitochondrial metabolism and co-ordinating specialized, pro-resolving mediators (SPMs), the melatonergic pathways, acetyl-coenzyme A, and the cyclooxygenase (COX) 2-prostaglandin (PG) E2 pathway that underpin 'exhaustion' in the endogenous anti-viral cells, paralleling similar metabolic suppression in cytolytic immune cells that is evident across all cancers. The pro-inflammatory cytokine induced gut permeability/dysbiosis and suppression of pineal melatonin are aspects of the wider pathophysiological underpinnings regulated by the AhR. This has a number of prophylactic and treatment implications for SARS-CoV-2 infection and cancers and future research directions that better investigate the biological underpinnings of social processes and how these may drive health disparities.

Melatonin: Roles in influenza, Covid-19, and other viral infections.

There is a growing appreciation that the regulation of the melatonergic pathways, both pineal and systemic, may be an important aspect in how viruses drive the cellular changes that underpin their control of cellular function. We review the melatonergic pathway role in viral infections, emphasizing influenza and covid-19 infections. Viral, or preexistent, suppression of pineal melatonin disinhibits neutrophil attraction, thereby contributing to an initial "cytokine storm", as well as the regulation of other immune cells. Melatonin induces the circadian gene, Bmal1, which disinhibits the pyruvate dehydrogenase complex (PDC), countering viral inhibition of Bmal1/PDC. PDC drives mitochondrial conversion of pyruvate to acetyl-coenzyme A (acetyl-CoA), thereby increasing the tricarboxylic acid cycle, oxidative phosphorylation, and ATP production. Pineal melatonin suppression attenuates this, preventing the circadian "resetting" of mitochondrial metabolism. This is especially relevant in immune cells, where shifting metabolism from glycolytic to oxidative phosphorylation, switches cells from reactive to quiescent phenotypes. Acetyl-CoA is a necessary cosubstrate for arylalkylamine N-acetyltransferase, providing an acetyl group to serotonin, and thereby initiating the melatonergic pathway. Consequently, pineal melatonin regulates mitochondrial melatonin and immune cell phenotype. Virus- and cytokine-storm-driven control of the pineal and mitochondrial melatonergic pathway therefore regulates immune responses. Virus-and cytokine storm-driven changes also increase gut permeability and dysbiosis, thereby suppressing levels of the short-chain fatty acid, butyrate, and increasing circulating lipopolysaccharide (LPS). The alterations in butyrate and LPS can promote viral replication and host symptom severity via impacts on the melatonergic pathway. Focussing on immune regulators has treatment implications for covid-19 and other viral infections.