Vaccinia-based oncolytic immunotherapy Pexastimogene Devacirepvec in patients with advanced hepatocellular carcinoma after sorafenib failure: a randomized multicenter Phase IIb trial (TRAVERSE).

Pexastimogene devacirepvec (Pexa-Vec) is a vaccinia virus-based oncolytic immunotherapy designed to preferentially replicate in and destroy tumor cells while stimulating anti-tumor immunity by expressing GM-CSF. An earlier randomized Phase IIa trial in predominantly sorafenib-naïve hepatocellular carcinoma (HCC) demonstrated an overall survival (OS) benefit. This randomized, open-label Phase IIb trial investigated whether Pexa-Vec plus Best Supportive Care (BSC) improved OS over BSC alone in HCC patients who failed sorafenib therapy (TRAVERSE). 129 patients were randomly assigned 2:1 to Pexa-Vec plus BSC vs. BSC alone. Pexa-Vec was given as a single intravenous (IV) infusion followed by up to 5 IT injections. The primary endpoint was OS. Secondary endpoints included overall response rate (RR), time to progression (TTP) and safety. A high drop-out rate in the control arm (63%) confounded assessment of response-based endpoints. Median OS (ITT) for Pexa-Vec plus BSC vs. BSC alone was 4.2 and 4.4 months, respectively (HR, 1.19, 95% CI: 0.78-1.80; p = .428). There was no difference between the two treatment arms in RR or TTP. Pexa-Vec was generally well-tolerated. The most frequent Grade 3 included pyrexia (8%) and hypotension (8%). Induction of immune responses to vaccinia antigens and HCC associated antigens were observed. Despite a tolerable safety profile and induction of T cell responses, Pexa-Vec did not improve OS as second-line therapy after sorafenib failure. The true potential of oncolytic viruses may lie in the treatment of patients with earlier disease stages which should be addressed in future studies. ClinicalTrials.gov: NCT01387555.

A serological survey of cattle in the Thames - Coromandel district of New Zealand for antibodies to Ross River virus.

AIM: To determine if cattle exposed to the southern saltmarsh mosquito (SSM), Aedes camptorhynchus, in the Thames-Coromandel district of New Zealand had been exposed to Ross River virus (RRV). METHODS: A purposive sampling design was used to test cattle from seven farms located in close proximity to four sites infested with A. camptorhynchus in the Thames-Coromandel district. Sera from 207 cattle were tested for antibodies to RRV, using an ELISA and confirmatory virus neutralisation test (VNT) as the gold standard. RESULTS: All 207 cattle tested negative for antibodies to RRV using the ELISA and VNT. CONCLUSIONS: This study found no evidence of exposure to RRV in cattle in locations in the Thames-Coromandel district of New Zealand where populations of SSM were present.

One-step, highly efficient site-directed mutagenesis by toxic protein selection.

A fast and efficient site-directed mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plasmid. Once the target gene is cloned into pTPS19, desired mutations can be introduced with two primers. The first contains the desired mutation, and the second is designed to create a +1 frame shift in the ccdB gene to inactivate the CcdB protein. The mutants can be directly selected on LB plates containing IPTG, through which the toxic CcdB protein is induced, thereby eliminating cells carrying wild-type parental plasmids. Based on stringent selection through the toxic CcdB protein, mutagenesis efficiency of 90%-100% was reached even after one round of transformation.

The structure and domain organization of Escherichia coli isocitrate lyase.

Enzymes of the glyoxylate-bypass pathway are potential targets for the control of many human diseases caused by such pathogens as Mycobacteria and Leishmania. Isocitrate lyase catalyses the first committed step in this pathway and the structure of this tetrameric enzyme from Escherichia coli has been determined at 2.1 A resolution. E. coli isocitrate lyase, like the enzyme from other prokaryotes, is located in the cytoplasm, whereas in plants, protozoa, algae and fungi this enzyme is found localized in glyoxysomes. Comparison of the structure of the prokaryotic isocitrate lyase with that from the eukaryote Aspergillus nidulans reveals a different domain structure following the deletion of approximately 100 residues from the larger eukaryotic enzyme. Despite this, the active sites of the prokaryotic and eukaryotic enzymes are very closely related, including the apparent disorder of two equivalent segments of the protein that are known to be involved in a conformational change as part of the enzyme's catalytic cycle.

A study of conserved in-loop and out-of-loop glycine residues in the large subunit of ribulose bisphosphate carboxylase/oxygenase by directed mutagenesis.

The replacement of all 22 completely conserved glycine residues in the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans by directed mutagenesis is described. In each beta/alpha barrel of the large subunit there are 12 completely conserved glycines in six of eight loops at the C-termini of eight beta-strands and four in loops at N-terminal ends of the beta-strands. Two completely conserved glycines are also in each beta/alpha barrel backbone and four more are in a large N-terminal portion preceding the barrel in a given L subunit. Substitution of glycines in loops that are C-terminal to beta-strands by proline was more deleterious to carboxylase activity than that by alanine supporting the postulates that these loops contribute to catalysis and substrate binding and that in some cases the glycines may serve as hinges enabling movement of the loops. In contrast, substitution of glycines at the N-terminal ends of beta-strands in the beta/alpha barrel more often led to failure to detect L subunits or their assembly into L8S8 complex. Substitution of these and the other conserved glycines by proline was more deleterious to carboxylase activity than by alanine in enzymes that assembled.

Lysine 194 is functional in isocitrate lyase from Escherichia coli.

Lysine 194 in conserved stretch 1 of tetrameric isocitrate lyase from Escherichia coli has been replaced by using the restriction-enzyme-site elimination method of directed mutagenesis. Expression of subunits of each variant and of wild-type (wt) enzyme was equivalent and all variants assembled into tetrameric proteins. The variants K194R and K194H had kcat values relative to that of wt enzyme taken as 100 of 11 and 7, respectively. Km values for Mg2+-Ds-isocitrate (in mM units) were: 0.13 for wt-enzyme; 0.12 for the K194R variant; and 0.55 for the K194H variant. Substitution at position 194 of Leu or Glu resulted in zero catalytic activity. These results establish that Lys 194 is another functional residue in conserved stretch one of isocitrate lyase from E. coli besides H184, K193, C195, and H197. Because K194 can be specifically replaced by the basic residues His and Arg with resultant lowered activity and by His with an increased Km value, it may contribute to a cation center and facilitate substrate binding as well as orientation of the developing transition state.

Activity and carboxylation specificity factor of mutant ribulose 1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans.

The values of molecular carboxylase activity kcat and carboxylation specificity factor tau for mutant ribulose 1,5-bisphosphate carboxylase (rubisco) from Anacystis nidulans decreased as compared to those of the wild type recombinant rubisco. The substitution of five amino acid residues in rubisco large subunit Lys,Ala,Ser,Thr,Leu(339-343)Phe,Leu,Met,Ile,Lys had kcat decreased by 90% and tau by 36.3%. The same parameters for mutants with the single replacements decreased: for Thr342Ile kcat by 40.5% and tau by 16.7%, and for mutant Leu343Lys kcat by 48.1% and tau by 18.5%. Mutant rubisco with three amino acid residues changed Val,Asp,Leu(346-348)Tyr,His,Thr was inactive. The substitution Leu326Ile decreased kcat by 54.4% and tau by 34.2%; and change Ser328Ala decreased kcat only by 5.6% but tau by 41.5%. Replacement Asn123His decreased kcat by 16.5%. Significance of the non conservative amino acid residues for carboxylase activity and ribulose-1,5-bisphosphate partition is discussed.

Serine319 and 321 are functional in isocitrate lyase from Escherichia coli.

With site-directed mutagenesis, Ser319 and Ser321 in conserved stretch 3 of tetrameric isocitrate lyase from Escherichiacoli were each substituted with alanine, cysteine, asparagine, or threonine in addition to simultaneous alanine/alanine substitutions. Besides their absolute conservation in all aligned isocitrate lyase sequences, the location of these serine residues, which flank a completely conserved proline, had been suggested in the active site in previous research by studies of photoinactivation of the enzyme by vanadate [Ko et al. (1992) J Biol Chem 267:91]. All substitutions for Ser321 and 319 except by threonine appreciably reduced the kcat of E. coli isocitrate lyase relative to that for wild-type (100) as follows: S319A, 0.4; S319C, 0.05; S319N, 0.01; S319T, 32.3; S321A, 2.9; S321C, 0.3; S321N, 0.1; S321T, 0.3; and S319A/S321A, 0, with little or no effect on the Km for the substrate Mg2+-Ds-isocitrate. The most active variant S319T exhibited threefold less activity than the wild-type enzyme; all variants assembled into tetramers. The S319T mutant isocitrate lyase was 100-fold more active than the S321T variant. This observation suggests that the requirement for a beta-hydroxymethyl group of serine in catalysis is less important at position 319 than at position 321. Although most singly substituted variants had very low isocitrate lyase activity, all variants harboring mutant isocitrate lyase of very low activity did grow on acetate asa sole carbon source albeit with longer doubling times and lag phases. Substitution of Pro320 by Ala, Asp, Gly, or His was highly detrimental to activity and increased the Km for substrate 3.5- to 8-fold; this suggests that Pro fixes the location of adjacent Ser OH groups and facilitates substrate binding and catalysis. From these collective results, it is proposed that Ser319 and Ser321 are involved in E.coli isocitrate lyase catalysis, perhaps by stabilizing the postulated reaction intermediate succinate trianion in the aci-carboxylate form and the related transition state via hydrogen bonding.

Sequence analysis of plasmid pCC5.2 from cyanobacterium Synechocystis PCC 6803 that replicates by a rolling circle mechanism.

The cyanobacterium Synechocystis sp. strain PCC 6803 contains several cryptic plasmids of 2.4, 5.2, and about 50 or 100 kbp. The complete nucleotide sequence of the 5.2-kbp plasmid, pCC5.2, has been analyzed and is reported here. This plasmid contains 5214 bp and 53.1% A+T. Six open reading frames, ORFs A-F (encoding peptides of larger than 90 amino acid residues), were located on both strands of pCC5.2. ORF B codes for a potential replication protein containing 971 amino acids, for which there are three homologous proteins encoded by other cyanobacterial plasmids. ORF C encodes a polypeptide of 93 amino acids which shares homologies with products of two ORFs found in the protein database. Counterparts of the products of ORF A, D, E, and F could not be found in the protein database. Detection of a single-stranded DNA intermediate during replication of pCC5.2 indicates that this plasmid may also replicate by a rolling circle mechanism, as has been reported for pCA2.4 and pCB2.4 from the same strain of Synechocystis (PCC 6803).

Cysteine 195 has a critical functional role in catalysis by isocitrate lyase from Escherichia coli.

Cysteine 195 in isocitrate lyase from Escherichia coli has been replaced by directed mutagenesis. Substitution by Ser yields enzyme with a k(cat) that is 0.03% that of wild type, and substitution by Ala, Gly, Thr, or Val yields completely inactive enzyme. The present results are consistent with a functional role of Cys 195.

The consequences of replacing histidine 356 in isocitrate lyase from Escherichia coli.

Isocitrate lyase from Escherichia coli has been expressed in transformed E. coli JE10 cells lacking the isocitrate lyase (icl) gene. After directed mutagenesis of icl by the restriction-site elimination method, partially purified isocitrate lyase mutants in which His 356 has been converted to Lys, Arg, Gln, Asp, or Leu have been characterized after induction of transformed, induced JE10 cells. Values of kcat compared to those for wild-type (wt) enzyme (100) at 37 degrees C, pH 7.3, are 18, 1, <1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively. Km values for the 1:1 Mg-isocitrate complex (in millimolar units) are: 0.13, wt; 0.11, H356K; and 0.63, H356R. Further chromatographic purification of isocitrate lyase yields highly purified wt, H356K, and H356R enzymes. The pH profile of the stability of isocitrate lyase, which has never been reported, showed that the H356R enzyme was unstable in the pH range investigated; the wt and H356R variant differed but each was sufficiently stable to study the pH dependence of catalysis. The log kcat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pKa and pKb values for dissociation of an ionizable group on the enzyme-substrate complex of <6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes. Plots of pKm vs pH were different for the wt and H356K variant. Values of pKa and pKb (derived from log kcat/Km plots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pKb value for the wt enzyme was 8.4. The data suggest that His 356 is an important functional residue in isocitrate lyase, perhaps in deprotonating isocitrate during catalytic cleavage.

A sensitive, simultaneous analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase efficiencies: Graphical determination of the CO2/O 2 specificity factor.

A simple approach to determine CO2/O2 specificity factor (τ) of ribulose 1,5-bisphosphate carboxylase/oxygenase is described. The assay measures the amount of CO2 fixation at varying [CO2]/[O2] ratios after complete consumption of ribulose 1,5-bisphosphate (RuBP). Carbon dioxide fixation catalyzed by the carboxylase was monitored by directly measuring the moles of (14)CO2 incorporated into 3-phosphoglycerate (PGA). This measurement at different [CO2]/[O2] ratios is used to determine graphically by several different linear plots the total RuBP consumed by the two activities and the CO2/O2 specificity factor. The assay can be used to measure the amounts of products of the carboxylase and oxygenase reactions and to determine the concentration of the substrate RuBP converted to an endpoint amount of PGA and phosphoglycolate. The assay was found to be suitable for all [CO2]/[O2] ratios examined, ranging from 14 to 215 micromolar CO2 (provided as 1-16 mM NaHCO3) and 614 micromolar O2 provided as 50% O2. The procedure described is extremely rapid and sensitive. Specificity factors for enzymes of highly divergent τ values are in good agreement with previously published data.

In vitro replication of cyanobacterial plasmids from Synechocystis PCC 6803.

Little knowledge of DNA replication in cyanobacteria is available. In this study, we report the development and characterization of an in vitro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803. This system (fraction III) was isolated at high salt concentrations and partially purified on a heparin-agarose column. DNA polymerases in Synechocystis 6803 appeared to be associated with membranes and could be released by the addition of ammonium sulfate to 20% saturation. DNA synthesis in fraction III was dependent on the addition of cyanobacterial plasmids isolated from the same strain. The in vitro replication products consist mostly of the supercoiled form of the plasmids. Unlike replication of many Escherichia coli plasmids, replication of cyanobacterial plasmids did not require added ATP, was not inhibited by omission of the ribonucleotides, and was insensitive to the RNA polymerase inhibitor rifampicin and the gyrase inhibitor novobiocin, but was inhibited by ethidium bromide. These data suggest that RNA may not be involved in the initiation of replication of cyanobacterial plasmids from Synechocystis 6803. In addition, intermediates of replication have been detected by two-dimensional gel electrophoresis. Density labeling experiments also indicate that cyanobacterial plasmid synthesis in vitro occurs by a semiconservative replication.

The complete DNA sequence and replication analysis of the plasmid pCB2.4 from the cyanobacterium Synechocystis PCC 6803.

Both strands of small plasmid pCB2.4 from the unicellular cyanobacterium Synechocystis PCC 6803 have been sequenced and analyzed. pCB2.4 contains 2345 bp and 57% A+T. Three open reading frames (ORFs) were identified in the nucleotide sequence of one strand of pCB2.4. ORF1 and ORF3 may encode basic proteins with calculated pI values of 7.71 and 9.96, respectively. The products of these ORFs have been compared to published protein sequences in databases and do not show significant homology to other proteins including replication proteins. pCB2.4 is not similar at the global DNA level to another 2.4-kbp plasmid, pCA2.4, present in the same cells, which has been found to replicate by a rolling-circle mechanism via a single-stranded intermediate. However, a short region (37 bp) is similar in pCA2.4 and pCB2.4. This region contains a potential nicking site (GATA) for replication proteins encoded by a group of plasmids that replicate by a rolling-circle mechanism and might be used as the origin of replication for these plasmids. Detection of a low level of single-stranded intermediate for pCB2.4 in the Synechocystis 6803 suggests that pCB2.4 may also replicate by a rolling-circle mechanism.

The importance of four histidine residues in isocitrate lyase from Escherichia coli.

By site-directed mutagenesis, substitutions were made for His-184 (H-184), H-197, H-266, and H-306 in Escherichia coli isocitrate lyase. Of these changes, only mutations of H-184 and H-197 appreciably reduced enzyme activity. Mutation of H-184 to Lys, Arg, or Leu resulted in an inactive isocitrate lyase, and mutation of H-184 to Gln resulted in an enzyme with 0.28% activity. Nondenaturing polyacrylamide gel electrophoresis demonstrated that isocitrate lyase containing the Lys, Arg, Gln, and Leu substitutions at H-184 was assembled poorly into the tetrameric subunit complex. Mutation of H-197 to Lys, Arg, Leu, and Gln resulted in an assembled enzyme with less than 0.25% wild-type activity. Five substitutions for H-266 (Asp, Glu, Val, Ser, and Lys), four substitutions for H-306 (Asp, Glu, Val, and Ser), and a variant in which both H-266 and H-306 were substituted for showed little or no effect on enzyme activity. All the H-197, H-266, and H-306 mutants supported the growth of isocitrate lyase-deficient E. coli JE10 on acetate as the sole carbon source; however, the H-184 mutants did not.

Active-site histidines in recombinant cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase examined by site-directed mutagenesis.

The functions of His(291), His(295) and His(324) at the active-site of recombinant A. nidulans ribulose-1,5-bisphosphate carboxylase/ oxygenase have been explored by site-directed mutagenesis. Replacement of His(291) by K or R resulted in unassembled proteins, while its replacement by E, Q or N resulted in assembled but inactive proteins. These results are in accord with a metal ion-binding role of this residue in the activated ternary complex by analogy to x-ray crystallographic analyses of tobacco and spinach enzymes.His(324) (H327 in spinach), which is located within bonding distance of the 5-phosphate of bound bi-substrate analog 2-carboxyarabinitol 1,5-bisphosphate in the crystal structures, has been substituted by A, K, R, Q and N. Again with the exception of the H324K and R variants, these changes resulted in detectable assembled protein. The mutant H324A protein exhibited no detectable carboxylase activity, whereas the H324Q and H324N changes resulted in purifiable holoenzyme with 2.0 and 0.1% of the recombinant wild-type specific carboxylase activity, respectively. These results are consistent with a phosphate binding role for this residue.The replacement of His(295), which has been suggested to aid in phosphate binding, with Ala in the A. nidulans enzyme leads to a mutant with 5.8% of the recombinant wild-type carboxylase activity. All other mutations at this position resulted in unassembled proteins. Purified H295A and H324Q enzymes had elevated Km(RuBP) values and unchanged CO2/O2 specificity factors compared to recombinant wild-type.

A small plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC 6803 encodes a rep protein and replicates by a rolling circle mechanism.

Different cryptic plasmids are widely distributed in many strains of cyanobacteria. A small cryptic plasmid, pCA2.4, from Synechocystis strain PCC 6803 was completely sequenced, and its replication mode was determined. pCA2.4 contained 2,378 bp and encoded a replication (Rep) protein, designated RepA. An analysis of the deduced amino acid sequence revealed that RepA of pCA2.4 has significant homology with Rep proteins of pKYM from Shigella sonnei, a pUB110 plasmid family from gram-positive bacteria, and with a protein corresponding to an open reading frame in a Nostoc plasmid and open reading frame C of Plectonema plasmid pRF1. pKYM and pUB110 family plasmids replicate by a rolling circle mechanism in which a Rep protein nicks the origin of replication to allow the generation of a single-stranded plasmid as a replication intermediate. RepA encoded by pC2.4 was expressed in Escherichia coli cells harboring a vector, pCRP336, containing the entire repA gene. The observed molecular weight of RepA was consistent with the value of 39,200 calculated from its deduced amino acid sequence, as was the N-terminal sequence analysis done through the 12th residue. Single-stranded plasmid DNA of pCA2.4 that was specifically degraded by S1 nuclease was detected in Synechocystis cells by Southern hybridization. These observations suggest that pCA2.4 replicates by a rolling circle mechanism in Synechocystis cells.