Heterologous Two-Dose Vaccination with Simian Adenovirus and Poxvirus Vectors Elicits Long-Lasting Cellular Immunity to Influenza Virus A in Healthy Adults.

BACKGROUND: T-cell responses against highly conserved influenza antigens have been previously associated with protection. However, these immune responses are poorly maintained following recovery from influenza infection and are not boosted by inactivated influenza vaccines. We have previously demonstrated the safety and immunogenicity of two viral vectored vaccines, modified vaccinia virus Ankara (MVA) and the chimpanzee adenovirus ChAdOx1 expressing conserved influenza virus antigens, nucleoprotein (NP) and matrix protein-1 (M1). We now report on the safety and long-term immunogenicity of multiple combination regimes of these vaccines in young and older adults. METHODS: We conducted a Phase I open-label, randomized, multi-center study in 49 subjects aged 18-46years and 24 subjects aged 50years or over. Following vaccination, adverse events were recorded and the kinetics of the T cell response determined at multiple time points for up to 18months. FINDINGS: Both vaccines were well tolerated. A two dose heterologous vaccination regimen significantly increased the magnitude of pre-existing T-cell responses to NP and M1 after both doses in young and older adults. The fold-increase and peak immune responses after a single MVA-NP+M1 vaccination was significantly higher compared to ChAdOx1 NP+M1. In a mixed regression model, T-cell responses over 18months were significantly higher following the two dose vaccination regimen of MVA/ChAdOx1 NP+M1. INTERPRETATION: A two dose heterologous vaccination regimen of MVA/ChAdOx1 NP+M1 was safe and immunogenic in young and older adults, offering a promising vaccination strategy for inducing long-term broadly cross-reactive protection against influenza A. FUNDING SOURCE: Medical Research Council UK, NIHR BMRC Oxford.

Xenopus phospho-CDK7/cyclin H expressed in baculoviral-infected insect cells.

The cyclin-dependent kinase-activating kinase (CAK) catalyzes the phosphorylation of the cyclin-dependent protein kinases (CDKs) on a threonine residue (Thr160 in human CDK2). The reaction is an obligatory step in the activation of the CDKs. In higher eukaryotes, the CAK complex has been characterized in two forms. The first consists of three subunits, namely CDK7, cyclin H, and an assembly factor called MAT1, while the second consists of phospho-CDK7 and cyclin H. Phosphorylation of CDK7 is essential for cyclin association and kinase activity in the absence of the assembly factor MAT1. The Xenopus laevis CDK7 phosphorylation sites are located on the activation segment of the kinase at residues Ser170 and at Thr176 (the latter residue corresponding to Thr160 in human CDK2). We report the expression and purification of X. laevis CDK7/cyclin H binary complex in insect cells through coinfection with the recombinant viruses, AcCDK7 and Accyclin H. Quantities suitable for crystallization trials have been obtained. The purified CDK7/cyclin H binary complex phosphorylated CDK2 and CDK2/cyclin A but did not phosphorylate histone H1 or peptide substrates based on the activation segments of CDK7 and CDK2. Analysis by mass spectrometry showed that coexpression of CDK7 with cyclin H in baculoviral-infected insect cells results in phosphorylation of residues Ser170 and Thr176 in CDK7. It is assumed that phosphorylation is promoted by kinase(s) in the insect cells that results in the correct, physiologically significant posttranslational modification. We discuss the occurrence of in vivo phosphorylation of proteins expressed in baculoviral-infected insect cells.

Osteodystrophia fibrosa in two guinea pigs.

Two adult guinea pigs were examined because they were lethargic and reluctant to walk. Additionally, I guinea pig had otitis media, and the other had dental malocclusion. Both guinea pigs had been fed a commercially available diet of cereals and pellets enriched with vitamin C and formulated for this species. Radiographically, the guinea pigs had coarse trabecular bone patterns, skeletal deformations, pathologic fractures, and polyarthritic degenerative joint disease. A double cortical line was also evident on several long bones, the pelvis, and the vertebrae. A diagnosis of osteopenia was confirmed by use of dual-energy x-ray absorptiometry. Analysis of a food sample fed to 1 guinea pig revealed calcium and phosphorus contents of 0.524 and 0.425%, respectively (Ca:P ratio, 1.23:1). Microscopic examination of bone tissue from both guinea pigs revealed severe fibrous osteodystrophy. Nutritional secondary hyperparathyroidism caused by calcium-phosphorus imbalance was considered to be the underlying cause of osteodystrophia fibrosa in both guinea pigs.

Identification of novel purine and pyrimidine cyclin-dependent kinase inhibitors with distinct molecular interactions and tumor cell growth inhibition profiles.

Substituted guanines and pyrimidines were tested as inhibitors of cyclin B1/CDK1 and cyclin A3/CDK2 and soaked into crystals of monomeric CDK2. O6-Cyclohexylmethylguanine (NU2058) was a competitive inhibitor of CDK1 and CDK2 with respect to ATP (Ki values: CDK1, 5 +/- 1 microM; CDK2, 12 +/- 3 microM) and formed a triplet of hydrogen bonds (i.e., NH-9 to Glu 81, N-3 to Leu 83, and 2-NH2 to Leu 83). The triplet of hydrogen bonding and CDK inhibition was reproduced by 2,6-diamino-4-cyclohexylmethyloxy-5-nitrosopyrimidine (NU6027, Ki values: CDK1, 2.5 +/- 0.4 microM; CDK2, 1.3 +/- 0.2 microM). Against human tumor cells, NU2058 and NU6027 were growth inhibitory in vitro (mean GI50 values of 13 +/- 7 microM and 10 +/- 6 microM, respectively), with a pattern of sensitivity distinct from flavopiridol and olomoucine. These CDK inhibition and chemosensitivity data indicate that the distinct mode of binding of NU2058 and NU6027 has direct consequences for enzyme and cell growth inhibition.

Activation of the particulate and not the soluble guanylate cyclase leads to the inhibition of Ca2+ extrusion through localized elevation of cGMP.

We examined whether localized increases in cytosolic cGMP have distinct regulatory effects on the concentration of cytosolic free Ca(2+) in ECV304 cells. Stimulation of the particulate guanylate cyclase by brain-type natriuretic peptide in fura-2-loaded cells caused a profound potentiation of the ATP-stimulated and thapsigargin-stimulated rise in cytosolic free Ca(2+). This effect is mediated by the inhibition of Ca(2+) extrusion via the plasma membrane Ca(2+)-ATPase pump. Furthermore, the addition of brain-type natriuretic peptide caused the partial inhibition of cation influx in ATP-stimulated cells. In contrast, elevation of cytosolic cGMP by activation of the soluble guanylate cyclase induced by the addition of sodium nitroprusside causes an increased reuptake of Ca(2+) into the intracellular stores without affecting cation influx or Ca(2+) efflux. Thus, localized pools of cGMP play distinct regulatory roles in the regulation of Ca(2+) homeostasis within individual cells. We define a new role for natriuretic peptides in the inhibition of Ca(2+) efflux that leads to the potentiation of agonist-evoked increases in cytosolic free Ca(2+).

Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity.

We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibits histone H1 kinase activity corresponding to approximately 0.3% of that observed with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements have shown that Thr160 phosphorylation increases the affinity of CDK2 for both histone substrate and ATP and decreases its affinity for ADP. By contrast, phosphorylation of CDK2 has a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 have been solved at 2.1-A resolution. The structures are similar, with the major difference occurring in the activation segment, which is disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystallization suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity observed.

Assembly of Amsacta moorei entomopoxvirus spheroidin into spheroids following synthesis in insect cells using a baculovirus vector.

The gene encoding the major occlusion body protein, spheroidin, of Amsacta moorei entomopoxvirus (AmEPV) was introduced into a baculovirus vector under control of the polyhedrin gene promoter. A recombinant virus produced large, ovoid occlusion body-like structures in both Spodoptera frugiperda and Trichoplusia ni cells. These structures resembled the spheroids found in AmEPV-infected Lymantria dispar cells, except they were devoid of virus particles and were not surrounded by a membrane- or envelope-like structure. These results were confirmed by immunofluoresence microscopy and Western blotting using a specific antipeptide antibody to spheroidin, and suggest that the supramolecular assembly of spheroids is not dependent on other EPV-encoded gene products. Transmission electron microscopy and subcellular fractionation experiments revealed that the spheroid-like structures were assembled in both the nucleus and cytoplasm of the recombinant virus-infected cells. This contrasts with the solely cytoplasmic localization found in AmEPV-infected cells.

Protein kinase inhibition by staurosporine revealed in details of the molecular interaction with CDK2.

Staurosporine exhibits nanomolar IC50 values against a wide range of protein kinases. The structure of a CDK2 staurosporine complex explains the tight binding of this inhibitor, and suggests features to be exploited in the design of specific inhibitors of CDKs.

Okadaic acid induces the release of Ca2+ from intracellular stores in ECV304 endothelial cells.

The effects of serine/threonine phosphatase inhibition on endothelial cell cytosolic free Ca2+ ([Ca2+]c) were investigated using okadaic acid and Fura-2-loaded ECV304 endothelial cells. When added to confluent adherent cells, 500 nM okadaic acid induced a transient and oscillatory elevation of [Ca2+]c both in the presence and absence of extracellular Ca2+. In the absence of extracellular Ca2+, depletion of the intracellular Ca2+ stores with either ATP (1 microM) or thapsigargin (100 nM) prevented any further release of Ca2+ on the subsequent addition of okadaic acid. Likewise (in the absence of extracellular Ca2+), a prior release of Ca2+ induced by okadaic acid reduced the magnitude of the response to ATP (1 microM). Taken together these observations indicate that okadaic acid induces Ca2+ release from the agonist-sensitive pool. The okadaic acid-induced Ca2+ release was mimicked by another potent phosphatase inhibitor, calyculin A (10 nM), and also the less potent analogue of okadaic acid, 1-nor-okadone (500 nM). The response to okadaic acid was characterised by a series of asynchronous [Ca2+]c oscillations, which at their peak resulted in 40-100% cells, at any one time, having an elevated [Ca2+]c. The response appeared to propagate between adjacent cells and the elevation of [Ca2+]c appeared initially in the cell periphery. In adherent cells, the release of Ca2+ induced by okadaic acid was found to be dependent upon cell density, as the proportion of cells responding to okadaic acid increased as the cell density increased. The response to okadaic acid was not observed in ECV304 cell suspensions. The data suggest that a kinase activity stimulated either directly or indirectly by cell-cell interactions can lead to the release of Ca2+ from the agonist- and thapsigargin-sensitive intracellular stores.

Modulation of mitochondrial Ca2+ in ECV304 endothelial cells by agents which elevate cAMP.

In the human umbilical vein endothelial cell-derived cell line, ECV304, we have previously shown that the elevation of [Ca2+]m in response to agonist stimulation is dependent on Ca2+ influx, i.e. an ATP-induced sustained increase in [Ca2+]c results in a slow-onset, sustained elevation in [Ca2+]m [Lawrie A.M., Rizzuto R., Pozzan T., Simpson A.W.M. A role for calcium influx in the regulation of mitochondrial calcium in endothelial cells. J Biol Chem 1996; 271: 10753-10759]. In this study, we have investigated the effect of raising cAMP on ATP-evoked elevations in both [Ca2+]m and [Ca2+]c by: (i) activating adenylate cyclase with the forskolin analogue--forskolin 6-[3'-(N,N-dimethylaminopropionyl)]-HCl (1 microM) (FA); (ii) addition of membrane permeable dibutyryl-cAMP (100 microM) (dbcAMP); and (iii) a combination of FA plus inhibition of cAMP phosphodiesterase using RO-20-1724 (17.5 microM) (RO);. We have found that protocols aimed at elevating cAMP significantly reduce the ATP-evoked (1-10 microM) rise in [Ca2+]m (n = 14); however, the [Ca2+]c response to ATP was not affected (n = 33). This new evidence shows that a second messenger system, other than Ca2+ itself, may influence [Ca2+]m changes in response to agonist stimulation.

A role for calcium influx in the regulation of mitochondrial calcium in endothelial cells.

By using an endothelial cell line (ECV304), derived from human umbilical vein and transfected with recombinant aequorin targeted to the mitochondrial matrix, we find that stimulation with ATP evokes long lasting increases in mitochondrial Ca2+ ([Ca2+]m) that largely depend on Ca2+ influx. In these cells, the release of stored Ca2+ is inefficient at elevating [Ca2+]m. Consequently it appears that in ECV304 cells, bulk cytosolic Ca2+ ([Ca2+]c) is the main determinant of [Ca2+]m changes. In ECV304 cells < 4% of mitochondria are within 700 nm of the endoplasmic reticulum as opposed to 65% in HeLa cells, whereas 14% are within 700 nm of the inner surface of the plasma membrane, compared with < 6% in HeLa cells. Following Ca2+ depletion, readdition of extracellular Ca2+ evokes an increase in [Ca2+]m but not in [Ca2+]c. Under these conditions, microdomains of high [Ca2+]c may occur beneath the plasma membrane of ECV304 cells resulting in the preferential elevation of Ca2+ in mitochondria located in this region. A model is discussed in which the localization of mitochondria with respect to Ca2+ sources is the main determinant of their in situ Ca2+ uptake kinetics. Thus, in any given cell type mitochondria may be localized to suit the energy and metabolic demands of their physiological actions.

High level synthesis and secretion of human urokinase using a late gene promoter of the Autographa californica nuclear polyhedrosis virus.

A cDNA encoding human urokinase was inserted into the Autographa californica nuclear polyhedrosis virus (AcNPV) genome at the polyhedrin gene locus under control of a duplicated copy of the late, basic protein gene promoter. The insect-derived urokinase was produced predominantly in the form of single-chain, pro-urokinase, with a molecular mass of 50 kDa, and demonstrated fibrinolytic activity. Synthesis and secretion of urokinase was first detected at 6 hours post-infection and continued steadily throughout the infection period. Comparisons with urokinase synthesised using the very late AcNPV polyhedrin gene promoter revealed that, although the polyhedrin promoter is intrinsically stronger, the yield of secreted urokinase was higher using the basic protein gene promoter. These data support the hypothesis that the host cell secretory pathway is compromised in the very late stages of baculovirus infection and may provide an explanation for why, in general, secreted and membrane-targeted proteins are not produced to the high levels observed with other proteins, when using very late baculovirus gene promoters.

Genetic modification of an entomopoxvirus: deletion of the spheroidin gene does not affect virus replication in vitro.

In the late stages of an entomopoxvirus infection, virions become embedded within a crystalline occlusion body or spheroid. Spheroids are composed primarily of a single polypeptide, spheroidin. We describe the construction of a genetically modified Amsacta moorei entomopoxvirus (AmEPV) in which the spheroidin gene coding sequences are deleted and replaced with those of a heterologous reporter gene encoding chloramphenicol acetyltransferase (CAT). A transfer vector, pAmCP1, was prepared containing a unique BamHI site in lieu of the spheroidin gene coding region, together with 1 kbp of upstream and downstream DNA sequence that flanks the spheroidin gene. The flanking sequences provide the transcriptional control signals and also guide homologous recombination so that the spheroidin gene coding region can be replaced with that of the foreign gene. The transfer vector was designed so that the translational start codon of the introduced foreign gene would be utilized. A recombinant virus, AmEPV.CAT, was produced by transfecting AmEPV-infected cells with the transfer vector encoding the CAT gene. The recombinant virus was isolated from wild-type virus by identifying plaques with a spheroidin-negative phenotype. Light microscopy and SDS-PAGE analysis demonstrated that no spheroids or spheroidin protein were produced in the recombinant virus-infected cells. The recombinant virus was able to replicate to high titres (10(7) p.f.u./ml) in insect cells indicating that the spheroidin gene is non-essential for AmEPV replication in vitro. Moderate levels of CAT were synthesized in recombinant virus-infected cells and temporal analyses indicated that CAT synthesis followed the pattern of spheroidin production suggesting that the spheroidin gene promoter was functioning under normal regulatory control in the genetically modified virus.

Ca2+ oscillations in pancreatic acinar cells: spatiotemporal relationships and functional implications.

The pancreatic acinar cells are of particular interest for the study of cytosolic Ca2+ signals, since they are morphologically polarized and generate agonist-specific Ca2+ oscillation patterns. Recent data obtained by combining digital video imaging of Fura-2 fluorescence with patch-clamp whole-cell current recording have provided new information on the spatiotemporal relationships of the cytosolic Ca2+ signals and the Ca(2+)-activated ionic currents. Low agonist concentrations evoke repetitive short-lasting local Ca2+ spikes in the secretory pole region that activate shortlasting current spikes. In the case of acetylcholine stimulation the spikes are confined to this region. When cholecystokinin is used the shortlasting local spikes precede longer Ca2+ transients that spread to the whole of the cell. Infusion of non-metabolizable inositol trisphosphate analogues can mimic these responses. The shortlasting local Ca2+ spikes are particularly sensitive to blockade by the inositol trisphosphate receptor antagonist heparin. These results show that the secretory pole region has a particularly high sensitivity to inositol trisphosphate probably due to clustering of high affinity receptors.

Two different spatiotemporal patterns for Ca2+ oscillations in pancreatic acinar cells: evidence of a role for protein kinase C in Ins(1,4,5)P3-mediated Ca2+ signalling.

The oscillations in cytosolic Ca2+ evoked in pancreatic exocrine acinar cells by submaximal concentrations of the two phosphoinositidase-coupled agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8) have very different temporal patterns. In the present study we use digital video imaging of Fura-2 fluorescence to map the spatial distribution of Ca2+ during the oscillating responses to these two agonists. The spatial patterns induced are very different for each of these agonists. ACh oscillations are sinusoidal and initiated at the secretory pole of these morphologically and functionally polarized cells. As they spread across the cell, pronounced gradients in Ca2+ develop that persist throughout the oscillating response. CCK-8 induces a series of discrete Ca2+ transients of longer duration and lower frequency. These elevations in Ca2+ arise slowly, throughout the cells and without any detectable gradients in Ca2+. We consider that the different spatiotemporal patterns can be explained on the basis of a physiologically relevant interaction between Ins(1,4,5)P3 and protein kinase C in second messenger-mediated Ca2+ signalling.

The pattern of agonist-evoked cytosolic Ca2+ oscillations depends on the resting intracellular Ca2+ concentration.

The agonists acetylcholine (ACh) and cholecystokinin (CCK) have been shown to evoke markedly different patterns of cytosolic Ca2+ oscillations in the same isolated pancreatic acinar cells. ACh induces high frequency sinusoidal oscillations (spiking) associated with activation of Ca2+ influx. CCK evokes longer lasting discrete transients separated by long intervals, and these low frequency transients persist for many minutes in the absence of extracellular Ca2+. Using digital imaging of fura-2 fluorescence, we have now monitored the free cytoplasmic Ca2+ concentration ([Ca2+]i) simultaneously in many individual cells from the same population. In the resting condition [Ca2+]i ranged from about 50 to 300 nM. When the resting [Ca2+]i was below 150 nM, ACh (50-100 nM) invariably evoked typical high frequency spiking. In the majority of cells which had a resting [Ca2+]i higher than 150 nM, ACh also evoked low frequency transients. Although initiated by ACh, these transients displayed the temporal and functional characteristics of the CCK-evoked transients. Removal of extracellular Ca2+ for a few minutes had no effect on this type of oscillation, whereas such a procedure reversibly abolished the ACh-evoked high frequency response. For the response evoked by 10-30 pM Ca2+ signal amplitude and the resting [Ca2+]i. Because the Ca2+ signal amplitude and the resting [Ca2+]i. Because CCK could never induce high frequency spiking there is some receptor specificity in dictating the time course of Ca2+ oscillations, but the resting [Ca2+]i is a major determinant of the Ca2+ signal pattern.

Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate.

Submaximal stimulation with agonists generating inositol 1,4,5-trisphosphate (IP3) evokes cytosolic Ca2+ oscillations in many different cell types. In general, each Ca2+ rise is initiated from a specific region near the plasma membrane and then spreads as a wave throughout the cell. We now demonstrate that low (physiological) agonist concentrations evoke local cytosolic Ca2+ spikes in the secretory pole of single mouse pancreatic acinar cells that are particularly sensitive to blockade by the IP3 receptor antagonist heparin. These spikes can occur alone or repetitively or can precede longer lasting Ca2+ signals that spread throughout the cell. Intracellular IP3 application mimics these agonist actions. The short-lasting local Ca2+ spikes provide an economical signaling mechanism and are of physiological significance since they activate Ca(2+)-dependent Cl- and cation currents important for control of fluid secretion.

Synchronous calcium oscillations in cerebellar granule cells in culture mediated by NMDA receptors.

The concentration of cytosolic Ca2+ ([Ca2+]i) was monitored in cerebellar granule cell cultures by digital imaging of fura-2 loaded cells. In the presence of Mg2+, cells grown in low K+ cultures responded to N-methyl-D-aspartate (NMDA) with uniform increases in [Ca2+]i from a stable basal [Ca2+]i. In contrast, in Mg(2+)-free medium, low K+ cultures showed spontaneous, synchronized [Ca2+]i oscillations from 4 days in culture. The oscillations were rapidly blocked by Mg2+, D,L-2-amino-5-phosphonovalerate, or tetrodotoxin. The development of oscillatory behaviour depended on the culture conditions and was not observed in cultures grown in high K+. These data show a high degree of connectivity established within 4 days in culture by dissociated granule cells allowing synchronized activity mediated through synaptic mechanisms.