A Novel Homozygous Mutation Causing Complete TYK2 Deficiency, with Severe Respiratory Viral Infections, EBV-Driven Lymphoma, and Jamestown Canyon Viral Encephalitis.

Autosomal recessive tyrosine kinase 2 (TYK2) deficiency is characterized by susceptibility to mycobacterial and viral infections. Here, we report a 4-year-old female with severe respiratory viral infections, EBV-driven Burkitt-like lymphoma, and infection with the neurotropic Jamestown Canyon virus. A novel, homozygous c.745C > T (p.R249*) variant was found in TYK2. The deleterious effects of the TYK2 lesion were confirmed by immunoblotting; by evaluating functional responses to IFN-α/ß, IL-10, and IL-23; and by assessing its scaffolding effect on the cell surface expression of cytokine receptor subunits. The effects of the mutation could not be pharmacologically circumvented in vitro, suggesting that alternative modalities, such as hematopoietic stem cell transplantation or gene therapy, may be needed. We characterize the first patient from Canada with a novel homozygous mutation in TYK2.

Real-world serological responses to extended-interval and heterologous COVID-19 mRNA vaccination in frail, older people (UNCoVER): an interim report from a prospective observational cohort study.

BACKGROUND: The use of COVID-19 vaccines has been prioritised to protect the most vulnerable-notably, older people. Because of fluctuations in vaccine availability, strategies such as delayed second dose and heterologous prime-boost have been used. However, the effectiveness of these strategies in frail, older people are unknown. We aimed to assess the antigenicity of mRNA-based COVID-19 vaccines in frail, older people in a real-world setting, with a rationed interval dosing of 16 weeks between the prime and boost doses. METHODS: This prospective observational cohort study was done across 12 long-term care facilities of the Montréal Centre-Sud - Integrated University Health and Social Services Centre in Montréal, Québec, Canada. Under a rationing strategy mandated by the provincial government, adults aged 65 years and older residing in long-term care facilities in Québec, Canada, with or without previously documented SARS-CoV-2 infection, were administered homologous or heterologous mRNA vaccines, with an extended 16-week interval between doses. All older residents in participating long-term care facilities who received two vaccine doses were eligible for inclusion in this study. Participants were enrolled from Dec 31, 2020, to Feb 16, 2021, and data were collected up to June 9, 2021. Clinical data and blood samples were serially collected from participants at the following timepoints: at baseline, before the first dose; 4 weeks after the first dose; 6-10 weeks after the first dose; 16 weeks after the first dose, up to 2 days before administration of the second dose; and 4 weeks after the second dose. Sera were tested for SARS-CoV-2-specific IgG antibodies (to the trimeric spike protein, the receptor-binding domain [RBD] of the spike protein, and the nucleocapsid protein) by automated chemiluminescent ELISA. Two cohorts were used in this study: a discovery cohort, for which blood samples were collected before administration of the first vaccine dose and longitudinally thereafter; and a confirmatory cohort, for which blood samples were only collected from 4 weeks after the prime dose. Analyses were done in the discovery cohort, with validation in the confirmatory cohort, when applicable. FINDINGS: The total study sample consisted of 185 participants. 65 participants received two doses of mRNA-1273 (Spikevax; Moderna), 36 received two doses of BNT162b2 (Comirnaty; Pfizer-BioNTech), and 84 received mRNA-1273 followed by BNT162b2. In the discovery cohort, after a significant increase in anti-RBD and anti-spike IgG concentrations 4 weeks after the prime dose (from 4·86 log binding antibody units [BAU]/mL to 8·53 log BAU/mL for anti-RBD IgG and from 5·21 log BAU/mL to 8·05 log BAU/mL for anti-spike IgG), there was a significant decline in anti-RBD and anti-spike IgG concentrations until the boost dose (7·10 log BAU/mL for anti-RBD IgG and 7·60 log BAU/mL for anti-spike IgG), followed by an increase 4 weeks later for both vaccines (9·58 log BAU/mL for anti-RBD IgG and 9·23 log BAU/mL for anti-spike IgG). SARS-CoV-2-naive individuals showed lower antibody responses than previously infected individuals at all timepoints tested up to 16 weeks after the prime dose, but achieved similar antibody responses to previously infected participants by 4 weeks after the second dose. Individuals primed with the BNT162b2 vaccine showed a larger decrease in mean anti-RBD and anti-spike IgG concentrations with a 16-week interval between doses (from 8·12 log BAU/mL to 4·25 log BAU/mL for anti-RBD IgG responses and from 8·18 log BAU/mL to 6·66 log BAU/mL for anti-spike IgG responses) than did those who received the mRNA-1273 vaccine (two doses of mRNA-1273: from 8·06 log BAU/mL to 7·49 log BAU/mL for anti-RBD IgG responses and from 6·82 log BAU/mL to 7·56 log BAU/mL for anti-spike IgG responses; mRNA-1273 followed by BNT162b2: from 8·83 log BAU/mL to 7·95 log BAU/mL for anti-RBD IgG responses and from 8·50 log BAU/mL to 7·97 log BAU/mL for anti-spike IgG responses). No differences in antibody responses 4 weeks after the second dose were noted between the two vaccines, in either homologous or heterologous combinations. INTERPRETATION: Interim results of this ongoing longitudinal study show that among frail, older people, previous SARS-CoV-2 infection and the type of mRNA vaccine influenced antibody responses when used with a 16-week interval between doses. In these cohorts of frail, older individuals with a similar age and comorbidity distribution, we found that serological responses were similar and clinically equivalent between the discovery and confirmatory cohorts. Homologous and heterologous use of mRNA vaccines was not associated with significant differences in antibody responses 4 weeks following the second dose, supporting their interchangeability. FUNDING: Public Health Agency of Canada, Vaccine Surveillance Reference Group; and the COVID-19 Immunity Task Force. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

RAGE and CCR7 mediate the transmigration of Zika-infected monocytes through the blood-brain barrier.

Details of the "Trojan Horse" mechanism by which Zika virus (ZIKV) crosses the blood-brain barrier (BBB) remain unclear. However, the migration of ZIKV-infected monocytes to the brain is thought to be dependent on both pattern-recognition and chemokine receptors. In this study, we investigated whether the migration of ZIKV-infected MonoMac-1 (MM-1) cells through the BBB is dependent on chemokine receptor 7 (CCR7) and receptor for advanced glycation end (RAGE); we also determined whether high mobility group box protein 1 (HMGB1) could facilitate the permeabilization of endothelial cells. We demonstrated that ZIKV infects MM-1 cells, leading to milieu accumulation of HMGB1. Our results suggest that HMGB1 is involved in the dysregulation of primary human brain microvascular endothelial cell junction markers. Our results also indicate that the migration of ZIKV-infected monocytes is dependent on chemokine ligand 19 (CCL19), the natural ligand of CCR7, in conditions recapitulating inflammation. RAGE-dependent migration of ZIKV-infected cells declined during transmigration assays in the presence of RAGE receptor antagonist FPS-ZM1. Understanding the molecular role of monocyte trafficking during ZIKV infections could facilitate the development of new therapeutic strategies to prevent the deleterious consequences of ZIKV neuroinfection.

TLR4 induces CCR7-dependent monocytes transmigration through the blood-brain barrier.

In this study, we examined whether bacterial pathogen-associated molecular patterns recognized by toll-like receptors (TLRs) can modify the CCR7-dependent migration of human monocytes. MonoMac-1 (MM-1) cells and freshly isolated human monocytes were cultivated in the presence of agonists for TLR4 (which senses lipopolysaccharides from gram-negative bacteria), TLR1/2 (which senses peptidoglycan from gram-positive bacteria), and TLR9 (which recognizes bacterial DNA rich in unmethylated CpG DNA). CCR7 mRNA transcription was measured using quantitative reverse transcription polymerase chain reaction and protein expression was examined using flow cytometry. CCR7 function was monitored using migration and transmigration assays in response to CCL19/CCL21, which are natural ligands for CCR7. Our results show that TLR4 strongly increases monocyte migratory capacity in response to CCL19 in chemotaxis and transmigration assays in a model that mimics the human blood-brain barrier, whereas TLR1/2 and 9 have no effect. Examination of monocyte migration in response to TLRs that are activated by bacterial components would contribute to understanding the excessive monocyte migration that characterizes the pathogenesis of bacterial infections and/or neuroinflammatory diseases.

CCR7 Receptor Expression in Mono-MAC-1 Cells: Modulation by Liver X Receptor α Activation and Prostaglandin E 2.

Cell migration via chemokine receptor CCR7 expression is an essential function of the immune system. We previously showed that prostaglandin E2 (PGE2), an important immunomodulatory molecule, increases CCR7 expression and function in monocytes. Here, we explore the role of the liver X receptor α (LXRα) activation on CCR7 expression in Mono-Mac-1 (MM-1) cells in the presence of PGE2. To do this, MM-1 cells were stimulated with the LXRα synthetic agonist T0901317 in the presence or absence of PGE2. CCR7 mRNA transcription was measured using quantitative RT-PCR and protein expression was examined using flow cytometry. CCR7 function was analyzed using migration assays in response to CCL19/CCL21, which are natural ligands for CCR7. Our results show that agonist-mediated activation of LXRα in the presence of PGE2 increases CCR7 mRNA transcription and MM-1 cell migratory capacity in response to CCL19/21. In addition, our results demonstrate that engagement of the E-prostanoids 2 and 4 (EP2/EP4) receptors present on MM-1 cells is responsible for the observed increase in CCR7 mRNA expression and function during LXRα activation. Examination of monocyte migration in response to lipid derivatives such as PGE2 and oxysterols that are produced at sites of chronic inflammation would contribute to understanding the excessive monocyte migration that characterizes atherosclerosis.

Kinetic and mechanistic characterization of Mycobacterium tuberculosis glutamyl-tRNA synthetase and determination of its oligomeric structure in solution.

Mycobacterium tuberculosis glutamyl-tRNA synthetase (Mt-GluRS), encoded by Rv2992c, was overproduced in Escherichia coli cells, and purified to homogeneity. It was found to be similar to the other well-characterized GluRS, especially the E. coli enzyme, with respect to the requirement for bound tRNA(Glu) to produce the glutamyl-AMP intermediate, and the steady-state kinetic parameters k(cat) (130 min(-1)) and K(M) for tRNA (0.7 microm) and ATP (78 microm), but to differ by a one order of magnitude higher K(M) value for L-Glu (2.7 mm). At variance with the E. coli enzyme, among the several compounds tested as inhibitors, only pyrophosphate and the glutamyl-AMP analog glutamol-AMP were effective, with K(i) values in the mum range. The observed inhibition patterns are consistent with a random binding of ATP and L-Glu to the enzyme-tRNA complex. Mt-GluRS, which is predicted by genome analysis to be of the non-discriminating type, was not toxic when overproduced in E. coli cells indicating that it does not catalyse the mischarging of E. coli tRNA(Gln) with L-Glu and that GluRS/tRNA(Gln) recognition is species specific. Mt-GluRS was significantly more sensitive than the E. coli form to tryptic and chymotryptic limited proteolysis. For both enzymes chymotrypsin-sensitive sites were found in the predicted tRNA stem contact domain next to the ATP binding site. Mt-GluRS, but not Ec-GluRS, was fully protected from proteolysis by ATP and glutamol-AMP. Small-angle X-ray scattering showed that, at variance with the E. coli enzyme that is strictly monomeric, the Mt-GluRS monomer is present in solution in equilibrium with the homodimer. The monomer prevails at low protein concentrations and is stabilized by ATP but not by glutamol-AMP. Inspection of small-angle X-ray scattering-based models of Mt-GluRS reveals that both the monomer and the dimer are catalytically active. By using affinity chromatography and His(6)-tagged forms of either GluRS or glutamyl-tRNA reductase as the bait it was shown that the M. tuberculosis proteins can form a complex, which may control the flux of Glu-tRNA(Glu) toward protein or tetrapyrrole biosynthesis.

Inhibition by L-aspartol adenylate of a nondiscriminating aspartyl-tRNA synthetase reveals differences between the interactions of its active site with tRNA(Asp) and tRNA(Asn).

Asparaginyl-tRNA formation in Pseudomonas aeruginosa PAO1 involves a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) which forms Asp-tRNA(Asp) and Asp-tRNA(Asn), and a tRNA-dependent amidotransferase which transamidates the latter into Asn-tRNA(Asn). We report here that the inhibition of this ND-AspRS by L-aspartol adenylate (Asp-ol-AMP), a stable analog of the natural reaction intermediate L-aspartyl adenylate, is biphasic because the aspartylation of the two tRNA substrates of ND-AspRS, tRNA(Asp) and tRNA(Asn), are inhibited with different Ki values (41 microM and 215 microM, respectively). These results reveal that the two tRNA substrates of ND-AspRS interact differently with its active site. Yeast tRNA(Asp) transcripts with some identity elements replaced by those of tRNA(Asn) have their aspartylation inhibited with Ki values different from that for the wild-type transcript. Therefore, aminoacyl adenylate analogs, which are competitive inhibitors of their cognate aminoacyl-tRNA synthetase, can be used to probe rapidly the role of various structural elements in positioning the tRNA acceptor end in the active site.

Synthesis of beta-ketophosphonate analogs of glutamyl and glutaminyl adenylate, and selective inhibition of the corresponding bacterial aminoacyl-tRNA synthetases.

The aminoacyl-beta-ketophosphonate-adenosines (aa-KPA) are stable analogs of the aminoacyl adenylates, which are high-energy intermediates in the formation of aminoacyl-tRNA catalyzed by aminoacyl-tRNA synthetases (aaRS). We have synthesized glutamyl-beta-ketophosphonate-adenosine (Glu-KPA) and glutaminyl-beta-ketophosphonate-adenosine (Gln-KPA), and have tested them as inhibitors of their cognate aaRS, and of a non-cognate aaRS. Glu-KPA is a competitive inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS) with a K(i) of 18microM with respect to its substrate glutamate, and binds at one site on this monomeric enzyme; the non-cognate Gln-KPA also binds this GluRS at one site, but is a much weaker (K(i)=2.9mM) competitive inhibitor. By contrast, Gln-KPA inhibits E. coli glutaminyl-tRNA synthetase (GlnRS) by binding competitively but weakly at two distinct sites on this enzyme (average K(i) of 0.65mM); the non-cognate Glu-KPA shows one-site weak (K(i)=2.8mM) competitive inhibition of GlnRS. These kinetic results indicate that the glutamine and the AMP modules of Gln-KPA, connected by the beta-ketophosphonate linker, cannot bind GlnRS simultaneously, and that one Gln-KPA molecule binds the AMP-binding site of GlnRS through its AMP module, whereas another Gln-KPA molecule binds the glutamine-binding site through its glutamine module. This model suggests that similar structural constraints could affect the binding of Glu-KPA to the active site of mammalian cytoplasmic GluRSs, which are evolutionarily much closer to bacterial GlnRS than to bacterial GluRS. This possibility was confirmed by the fact that Glu-KPA inhibits bovine liver GluRS 145-fold less efficiently than E. coli GluRS by competitive weak binding at two distinct sites (average K(i)=2.6mM). Moreover, these kinetic differences reveal that the active sites of bacterial GluRSs and mammalian cytoplasmic GluRSs have substantial structural differences that could be further exploited for the design of better inhibitors specific for bacterial GluRSs, promising targets for antimicrobial therapy.

Structural bases of transfer RNA-dependent amino acid recognition and activation by glutamyl-tRNA synthetase.

Glutamyl-tRNA synthetase (GluRS) is one of the aminoacyl-tRNA synthetases that require the cognate tRNA for specific amino acid recognition and activation. We analyzed the role of tRNA in amino acid recognition by crystallography. In the GluRS*tRNA(Glu)*Glu structure, GluRS and tRNA(Glu) collaborate to form a highly complementary L-glutamate-binding site. This collaborative site is functional, as it is formed in the same manner in pretransition-state mimic, GluRS*tRNA(Glu)*ATP*Eol (a glutamate analog), and posttransition-state mimic, GluRS*tRNA(Glu)*ESA (a glutamyl-adenylate analog) structures. In contrast, in the GluRS*Glu structure, only GluRS forms the amino acid-binding site, which is defective and accounts for the binding of incorrect amino acids, such as D-glutamate and L-glutamine. Therefore, tRNA(Glu) is essential for formation of the completely functional binding site for L-glutamate. These structures, together with our previously described structures, reveal that tRNA plays a crucial role in accurate positioning of both L-glutamate and ATP, thus driving the amino acid activation.

Glutamylsulfamoyladenosine and pyroglutamylsulfamoyladenosine are competitive inhibitors of E. coli glutamyl-tRNA synthetase.

5'-O-[N-(L-glutamyl)-sulfamoyl] adenosine is a potent competitive inhibitor of E. coli glutamyl-tRNA synthetase with respect to glutamic acid (K(i) = 2.8 nM) and is the best inhibitor of this enzyme. It is a weaker inhibitor of mammalian glutamyl-tRNA synthetase (K(i) = 70 nM). The corresponding 5'-O-[N-(L-pyroglutamyl)-sulfamoyl] adenosine is a weak inhibitor (K(i) = 15 microM) of the E. coli enzyme.

Synthesis and aminoacyl-tRNA synthetase inhibitory activity of aspartyl adenylate analogs.

Three nonhydrolyzable aspartyl adenylate analogs have been prepared and tested as inhibitors of E. coli aspartyl-tRNA synthetase. 5'-O-[N-(L-Aspartyl)sulfamoyl]adenosine is a potent competitive inhibitor (K(i) = 15 nM) whereas L-aspartol adenylate is a weaker inhibitor (K(i) = 45 microM) with respect to aspartic acid. The corresponding ketomethylphosphonate (a novel isosteric replacement) is also a strong inhibitor (K(i) = 123 nM).

ATP binding by glutamyl-tRNA synthetase is switched to the productive mode by tRNA binding.

Aminoacyl-tRNA synthetases catalyze the formation of an aminoacyl-AMP from an amino acid and ATP, prior to the aminoacyl transfer to tRNA. A subset of aminoacyl-tRNA synthetases, including glutamyl-tRNA synthetase (GluRS), have a regulation mechanism to avoid aminoacyl-AMP formation in the absence of tRNA. In this study, we determined the crystal structure of the 'non-productive' complex of Thermus thermophilus GluRS, ATP and L-glutamate, together with those of the GluRS.ATP, GluRS.tRNA.ATP and GluRS.tRNA.GoA (a glutamyl-AMP analog) complexes. In the absence of tRNA(Glu), ATP is accommodated in a 'non-productive' subsite within the ATP-binding site, so that the ATP alpha-phosphate and the glutamate alpha-carboxyl groups in GluRS. ATP.Glu are too far from each other (6.2 A) to react. In contrast, the ATP-binding mode in GluRS.tRNA. ATP is dramatically different from those in GluRS.ATP.Glu and GluRS.ATP, but corresponds to the AMP moiety binding mode in GluRS.tRNA.GoA (the 'productive' subsite). Therefore, tRNA binding to GluRS switches the ATP-binding mode. The interactions of the three tRNA(Glu) regions with GluRS cause conformational changes around the ATP-binding site, and allow ATP to bind to the 'productive' subsite.