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There is widespread interest in facile methods for generating potent neutralizing antibodies, nanobodies, and other affinity proteins against SARS-CoV-2 and related viruses to address current and future pandemics. While isolating antibodies from animals and humans are proven approaches, these methods are limited to the affinities, specificities, and functional activities of antibodies generated by the immune system. Here we report a surprisingly simple directed evolution method for generating nanobodies with high affinities and neutralization activities against SARS-CoV-2. We demonstrate that complementarity-determining region swapping between low-affinity lead nanobodies, which we discovered unintentionally but find is simple to implement systematically, results in matured nanobodies with unusually large increases in affinity. Importantly, the matured nanobodies potently neutralize both SARS-CoV-2 pseudovirus and live virus, and possess drug-like biophysical properties. We expect that our methods will improve in vitro nanobody discovery and accelerate the generation of potent neutralizing nanobodies against diverse coronaviruses., Graphical abstract, Zupancic et al. report potent neutralizing nanobodies against SARS-CoV-2. They demonstrate an approach that involves swapping the complementarity-determining regions of low-affinity clones to generate matured nanobodies with large increases in affinity and neutralization activity.

Graphical abstract, Adenovirus-associated virus is a powerful vector system for transducing cells in vivo. It is widely used in animal systems due to high transduction efficiency of non-dividing cells with more than a dozen serotypes that have preferential tissue tropism. The viral genome remains episomal in the nucleus but maintains sustained expression in terminally differentiated cells for several weeks to months. Despite the popularity of recombinant AAV (rAAV) vectors, quality control testing of the virus after production is largely limited to physical characteristics such as viral genomes/ml determinations and silver staining acrylamide gels to determine purity. Functional testing, in vivo, is not practical due to high cost and restricted access of animal care and long duration of the assay (2–3 weeks). Some functional testing can be accomplished in cultured cells such as HEK293 cells, but HEK293 cells limit the types of rAAV constructs that can be tested. Many rAAV constructs are designed to study neurons in the brain with neural-specific promoters and many are floxed with loxp sites to be “activated” only in Cre-expressing neurons in transgenic animals. To develop a reporter cell line for rapid rAAV quality control assessment of these neural-specific, floxed rAAV constructs, we used the lentiviral system to stably express Cre recombinase in the SH-SY5Y neuroblastoma cell line. • A simple and economic method to evaluate recombinant AAV in vitro. • Allows functional validation of rAAV across a wide range of serotypes and promoters. • Allows functional validation of Cre-dependent rAAV constructs.

Graphical abstract, PCR is a powerful tool for generating specific fragments of DNA that can be used to create gene variations or tagged expression constructs. Overlap extension PCR is a valuable technique that is commonly used for cloning large complex fragments, making edits to cloned genes or fusing two gene elements together. After difficulties in utilizing this technique following existing methods, we developed an optimized protocol. To accomplish this, three significant changes were made; 1) touchdown PCR cycling parameters were used to eliminate the need for optimizing PCR cycling conditions, 2) the high-fidelity, high-processivity Q5 DNA polymerase was used to improve full-length amplification quality, and 3) a reduced amount of primer in the final PCR amplification step decreased non-specific amplimers. This modified protocol results in consistent generation of gene fusion products, with little to no background and enhanced efficiency of the transgene construction process.

Thyroid C-cell lines, such as the CA77, 44-2C, and TT lines, are derived from calcitonin-secreting endocrine cells of neural crest origin. An intriguing property of C-cells is that they possess neuronal properties. These include neurite extension and expression of neuronal markers including neurofilaments, the neuropeptide calcitonin gene-related peptide (CGRP), and Ca2+ and Na+ currents characteristic of neurons. These phenotypes can be enhanced or repressed by various agents and substrata. Most remarkably, C-cells also possess numerous characteristics of serotonergic neurons, including expression of tryptophan hydroxylase and a fluoxetine-inhibitable reuptake transporter. These properties suggest a role for the use of C-cell lines as neuronal model systems. We will describe the use of these cell lines in examining the role of exogenous factors in neural differentiation and as a source of cDNA for identification of differentially expressed genes. In addition, the serotonergic properties of C-cell lines will be explored, along with techniques specific to their use as serotonergic model systems. Lastly, the use of the CA77 and 44-2C C-cell lines in analysis of cis- and trans-acting transcription factors is exemplified using the calcitonin/CGRP gene. Thus, C-cell lines offer significant potential as models for cellular and molecular analyses of peptidergic and serotonergic neurons.

We have isolated an alternative transcript of the rat Gs alpha signal transduction protein gene, referred to as Gs alpha N1. Gs alpha N1 was isolated by differential hybridization screening of genes induced upon dexamethasone treatment of the neuronal-like CA77 rat thyroid C-cell line. The 1-kilobase Gs alpha N1 transcript is generated by alternative splicing and polyadenylation of a novel terminal exon. This exon lies 800 base pairs downstream of exon 3 in the Gs alpha gene. Dexamethasone differentially induced Gs alpha N1 severalfold relative to Gs alpha mRNA in the CA77 cells, similar to the bias seen with alternative processing of the calcitonin/calcitonin gene-related peptide transcript. In addition to the differential regulation by dexamethasone, the expression pattern of Gs alpha N1 in rat tissues differed markedly from Gs alpha. Gs alpha N1 mRNA was much more abundant in the brain, with intermediate levels in skeletal muscle and very low levels in other tissues. This was in contrast to the more ubiquitously expressed Gs alpha mRNA. Within the brain, Gs alpha N1 was particularly abundant in discrete regions of the brainstem and hypothalamus that modulate autonomic functions. Examination of rat embryos demonstrated that Gs alpha is expressed in both brain and nonneural tissue at least 1 day before Gs alpha N1 mRNA could be detected in the embryonic brain. Based on the regulated expression of the Gs alpha N1 transcript and previous studies on G alpha proteins, the predicted Gs alpha N1 protein may potentially modulate several heterotrimeric G protein functions in the nervous system.