Clinical Validation of a Novel T-Cell Receptor Sequencing Assay for Identification of Recent or Prior Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BACKGROUND: While diagnostic, therapeutic, and vaccine development in the coronavirus disease 2019 (COVID-19) pandemic has proceeded at unprecedented speed, critical gaps in our understanding of the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unaddressed by current diagnostic strategies. METHODS: A statistical classifier for identifying prior SARS-CoV-2 infection was trained using >4000 SARS-CoV-2-associated T-cell receptor (TCR) ß sequences identified by comparing 784 cases and 2447 controls from 5 independent cohorts. The T-Detect COVID (Adaptive Biotechnologies) assay applies this classifier to TCR repertoires sequenced from blood samples to yield a binary assessment of past infection. Assay performance was assessed in 2 retrospective (n = 346; n = 69) and 1 prospective cohort (n = 87) to determine positive percent agreement (PPA) and negative percent agreement (NPA). PPA was compared with 2 commercial serology assays, and pathogen cross-reactivity was evaluated. RESULTS: T-Detect COVID demonstrated high PPA in individuals with prior reverse transcription-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infection (97.1% 15+ days from diagnosis; 94.5% 15+ days from symptom onset), high NPA (∼100%) in presumed or confirmed SARS-CoV-2 negative cases, equivalent or higher PPA than 2 commercial serology tests, and no evidence of pathogen cross-reactivity. CONCLUSIONS: T-Detect COVID is a novel T-cell immunosequencing assay demonstrating high clinical performance for identification of recent or prior SARS-CoV-2 infection from blood samples, with implications for clinical management, risk stratification, surveillance, and understanding of protective immunity and long-term sequelae.

Apicoplast targeting of a Toxoplasma gondii transmembrane protein requires a cytosolic tyrosine-based motif.

Toxoplasma gondii, like most apicomplexan parasites, possesses an essential relict chloroplast, the apicoplast. Several apicoplast membrane proteins lack the bipartite targeting sequences of luminal proteins. Vesicles bearing these membrane proteins are detected during apicoplast enlargement, but the means of cargo selection remains obscure. We used a combination of deletion mutagenesis, point mutations and protein chimeras to identify a short motif prior to the first transmembrane domain of the T. gondii apicoplast phosphate transporter 1 (APT1) that is necessary for apicoplast trafficking. Tyrosine 16 was essential for proper localization; any substitution resulted in misdirection of APT1 to the Golgi body. Glycine 17 was also important, with significant Golgi body accumulation in the alanine mutant. Separation of at least eight amino acids from the transmembrane domain was required for full motif function. Similarly placed YG motifs are present in apicomplexan APT1 orthologs and the corresponding N-terminal domain from Plasmodium vivax was able to route T. gondii APT1 to the apicoplast. Differential permeabilization showed that both the N- and C-termini of APT1 are exposed to the cytosol. We propose that this YG motif facilitates APT1 trafficking via interactions that occur on the cytosolic face of nascent vesicles destined for the apicoplast.

Unusual features and localization of the membrane kinome of Trypanosoma brucei.

In many eukaryotes, multiple protein kinases are situated in the plasma membrane where they respond to extracellular ligands. Ligand binding elicits a signal that is transmitted across the membrane, leading to activation of the cytosolic kinase domain. Humans have over 100 receptor protein kinases. In contrast, our search of the Trypanosoma brucei kinome showed that there were only ten protein kinases with predicted transmembrane domains, and unlike other eukaryotic transmembrane kinases, seven are predicted to bear multiple transmembrane domains. Most of the ten kinases, including their transmembrane domains, are conserved in both Trypanosoma cruzi and Leishmania species. Several possess accessory domains, such as Kelch, nucleotide cyclase, and forkhead-associated domains. Surprisingly, two contain multiple regions with predicted structural similarity to domains in bacterial signaling proteins. A few of the protein kinases have previously been localized to subcellular structures such as endosomes or lipid bodies. We examined the localization of epitope-tagged versions of seven of the predicted transmembrane kinases in T. brucei bloodstream forms and show that five localized to the endoplasmic reticulum. The last two kinases are enzymatically active, integral membrane proteins associated with the flagellum, flagellar pocket, or adjacent structures as shown by both fluorescence and immunoelectron microscopy. Thus, these kinases are positioned in structures suggesting participation in signal transduction from the external environment.