Evaluation of the safety, reactogenicity and immunogenicity of FluBlok® trivalent recombinant baculovirus-expressed hemagglutinin influenza vaccine administered intramuscularly to healthy adults 50-64 years of age.

BACKGROUND: Alternative methods for influenza vaccine production are needed to ensure adequate supplies. METHODS: Healthy adults 50-64 years were assigned randomly to receive one intramuscular injection of trivalent recombinant hemagglutinin (rHA) or U.S. licensed trivalent inactivated vaccine (TIV) containing H1, H3 and B antigens (Ag) derived from 2007 to 2008 influenza virus strains A/Solomon Islands/03/2006 (H1N1), A/Wisconsin/67/2005 (H3N2), and B/Malaysia/2506/2004. Each rHA dose contained 45 µg HA/strain of the 2007-2008 FDA-recommended Ag vs. 15 µg/strain for TIV. Antibody (Ab) responses were measured using a hemagglutination-inhibition (HAI) assay at baseline and 28 days post-vaccination. Respiratory samples for viral culture were collected from subjects with influenza-like illness (ILI) during the 2007-2008 season in the U.S. RESULTS: 601 subjects were enrolled. Vaccines were well tolerated. Seroconversion (the percentage of subjects with either (a) a pre-vaccination HAI titer ≤ 10 and a post-vaccination HAI titer ≥ 40 or (b) a pre-vaccination titer ≥ 10 and a minimum four-fold rise in post-vaccination HAI antibody titer) in the TIV and rHA groups, respectively, was obtained in 66% vs. 72% for H1; 44% vs. 61% for H3; and 41% vs. 41% for B. Proportions achieving titers ≥ 40 were 96% vs. 96% for H1, 75% vs. 85% for H3, and 94% vs. 93% vs. B. Geometric mean titer ratios at day 28 (TIV/rHA) were 0.77 for H1; 0.58 for H3; and 1.05 for B, respectively. ILI frequencies were low and similar in both groups. CONCLUSIONS: Both vaccines were safe and immunogenic. Ab responses vs. H1 and H3 Ags were significantly higher in the rHA group, with similar responses to B. Furthermore, the FluBlok group had a statistically significantly higher seroconversion rate against influenza A/H3N2 compared to the TIV group.

Comparative immunogenicity of recombinant influenza hemagglutinin (rHA) and trivalent inactivated vaccine (TIV) among persons > or =65 years old.

Alternative substrates for influenza vaccine production are needed to ensure adequate supplies. We evaluated the relative safety and immunogenicity of recombinant hemagglutinin (rHA) or trivalent inactivated vaccine (TIV) among 869 > or =65-year-old subjects in a randomized clinical trial. Virologic surveillance for influenza-like illness (ILI) was conducted during the 2006-2007 epidemic. Vaccines were well tolerated. Seroconversion rates vs. influenza A/H1N1 and H3N2 antigens were superior in the rHA group, but were inferior vs. influenza B; however, results for influenza B are confounded since the vaccine antigens were different. ILI frequencies were low and similar in both groups. Studies assessing relative immunogenicity of vaccines using identical B Ags are warranted.

Neurobehavioral abnormalities in the dysbindin-1 mutant, sandy, on a C57BL/6J genetic background.

Sandy mice have a deletion mutation in the gene encoding dysbindin-1, Dtnbp1, with consequent reduction of the protein in heterozygotes and its loss in homozygotes. The sandy mouse thus serves as an animal model of dysbindin-1 function. As this protein is concentrated in synaptic tissue and affects transmitter release, it may affect neuronal processes that mediate behavior. To investigate the neurobehavioral effects of the Dtnbp1 mutation, we studied littermate sandy and wild-type controls on a C57BL/6J genetic background. The three animal groups were indistinguishable in their external physical characteristics, sensorimotor skills and indices of anxiety-like behaviors. In the open field, however, homozygous animals were hyperactive and appeared to show less habituation to the initially novel environment. In the Morris water maze, homozygous animals displayed clear deficits in spatial learning and memory with marginal deficits in visual association learning. Apart from the last mentioned deficits, these abnormalities are consistent with hippocampal dysfunction and in some cases with elevated dopaminergic transmission via D2 dopamine receptors. As similar deficits in spatial learning and memory have been found in schizophrenia, where decreased dysbindin-1 has been found in the hippocampus, the sandy mouse may also model certain aspects of cognition and behavior relevant to schizophrenia.

Vaccines in development against avian influenza.

Avian influenza has become a high priority item for all public health authorities. An influenza pandemic is believed to be imminent. The only questions are what will be the causative agent and when will it happen. Recently, most attention has been directed to human cases of avian influenza caused by an H5N1 avian influenza virus. An effective vaccine will be needed to substantially reduce the impact of an influenza pandemic. Despite the fact that the current influenza vaccine manufacturing technology is not adequate to support vaccine production in the event of a pandemic influenza outbreak, most of the ongoing clinical development is occurring with vaccines made in embryonated chicken eggs. It is clear that innovative production technology is required. This review provides an update on the status of avian vaccine development. In addition available manufacturing technologies are presented, some of which could be more suitable to adequately respond to an emergency situation where billions of doses of vaccines would be required within a very short period of time. The review is concluded with some proposed areas of focus for pandemic vaccine preparedness.

The RecOR proteins modulate RecA protein function at 5' ends of single-stranded DNA.

The Escherichia coli RecF, RecO and RecR pro teins have previously been implicated in bacterial recombinational DNA repair at DNA gaps. The RecOR-facilitated binding of RecA protein to single-stranded DNA (ssDNA) that is bound by single-stranded DNA-binding protein (SSB) is much faster if the ssDNA is linear, suggesting that a DNA end (rather than a gap) facilitates binding. In addition, the RecOR complex facilitates RecA protein-mediated D-loop formation at the 5' ends of linear ssDNAs. RecR protein remains associated with the RecA filament and its continued presence is required to prevent filament disassembly. RecF protein competes with RecO protein for RecR protein association and its addition destabilizes RecAOR filaments. An enhanced function of the RecO and RecR proteins can thus be seen in vitro at the 5' ends of linear ssDNA that is not as evident in DNA gaps. This function is countered by the RecF/RecO competition for association with the RecR protein.

Recombinational DNA repair of damaged replication forks in Escherichia coli: questions.

It has recently become clear that the recombinational repair of stalled replication forks is the primary function of homologous recombination systems in bacteria. In spite of the rapid progress in many related lines of inquiry that have converged to support this view, much remains to be done. This review focuses on several key gaps in understanding. Insufficient data currently exists on: (a) the levels and types of DNA damage present as a function of growth conditions, (b) which types of damage and other barriers actually halt replication, (c) the structures of the stalled/collapsed replication forks, (d) the number of recombinational repair paths available and their mechanistic details, (e) the enzymology of some of the key reactions required for repair, (f) the role of certain recombination proteins that have not yet been studied, and (g) the molecular origin of certain in vivo observations associated with recombinational DNA repair during the SOS response. The current status of each of these topics is reviewed.

RecA protein filaments disassemble in the 5' to 3' direction on single-stranded DNA.

RecA protein forms filaments on both single- and double-stranded DNA. Several studies confirm that filament extension occurs in the 5' to 3' direction on single-stranded DNA. These filaments also disassemble in an end-dependent fashion, and several indirect observations suggest that the disassembly occurs on the end opposite to that at which assembly occurs. By labeling the 5' end of single-stranded DNA with a segment of duplex DNA, we demonstrate unambiguously that RecA filaments disassemble uniquely in the 5' to 3' direction.

DNA pairing and strand exchange by the Escherichia coli RecA and yeast Rad51 proteins without ATP hydrolysis: on the importance of not getting stuck.

The bacterial RecA protein and the homologous Rad51 protein in eukaryotes both bind to single-stranded DNA (ssDNA), align it with a homologous duplex, and promote an extensive strand exchange between them. Both reactions have properties, including a tolerance of base analog substitutions that tend to eliminate major groove hydrogen bonding potential, that suggest a common molecular process underlies the DNA strand exchange promoted by RecA and Rad51. However, optimal conditions for the DNA pairing and DNA strand exchange reactions promoted by the RecA and Rad51 proteins in vitro are substantially different. When conditions are optimized independently for both proteins, RecA promotes DNA pairing reactions with short oligonucleotides at a faster rate than Rad51. For both proteins, conditions that improve DNA pairing can inhibit extensive DNA strand exchange reactions in the absence of ATP hydrolysis. Extensive strand exchange requires a spooling of duplex DNA into a recombinase-ssDNA complex, a process that can be halted by any interaction elsewhere on the same duplex that restricts free rotation of the duplex and/or complex, I.e. the reaction can get stuck. Optimization of an extensive DNA strand exchange without ATP hydrolysis requires conditions that decrease nonproductive interactions of recombinase-ssDNA complexes with the duplex DNA substrate.

Historical overview: searching for replication help in all of the rec places.

For several decades, research into the mechanisms of genetic recombination proceeded without a complete understanding of its cellular function or its place in DNA metabolism. Many lines of research recently have coalesced to reveal a thorough integration of most aspects of DNA metabolism, including recombination. In bacteria, the primary function of homologous genetic recombination is the repair of stalled or collapsed replication forks. Recombinational DNA repair of replication forks is a surprisingly common process, even under normal growth conditions. The new results feature multiple pathways for repair and the involvement of many enzymatic systems. The long-recognized integration of replication and recombination in the DNA metabolism of bacteriophage T4 has moved into the spotlight with its clear mechanistic precedents. In eukaryotes, a similar integration of replication and recombination is seen in meiotic recombination as well as in the repair of replication forks and double-strand breaks generated by environmental abuse. Basic mechanisms for replication fork repair can now inform continued research into other aspects of recombination. This overview attempts to trace the history of the search for recombination function in bacteria and their bacteriophages, as well as some of the parallel paths taken in eukaryotic recombination research.

RecA protein promotes the regression of stalled replication forks in vitro.

Replication forks are halted by many types of DNA damage. At the site of a leading-strand DNA lesion, forks may stall and leave the lesion in a single-strand gap. Fork regression is the first step in several proposed pathways that permit repair without generating a double-strand break. Using model DNA substrates designed to mimic one of the known structures of a fork stalled at a leading-strand lesion, we show here that RecA protein of Escherichia coli will promote a fork regression reaction in vitro. The regression process exhibits an absolute requirement for ATP hydrolysis and is enhanced when dATP replaces ATP. The reaction is not affected by the inclusion of the RecO and R proteins. We present this reaction as one of several potential RecA protein roles in the repair of stalled and/or collapsed replication forks in bacteria.

Product analysis illuminates the final steps of IES deletion in Tetrahymena thermophila.

DNA sequences (IES elements) eliminated from the developing macronucleus in the ciliate Tetrahymena thermophila are released as linear fragments, which have now been detected and isolated. A PCR-mediated examination of fragment end structures reveals three types of strand scission events, reflecting three steps in the deletion process. New evidence is provided for two steps proposed previously: an initiating double-stranded cleavage, and strand transfer to create a branched deletion intermediate. The fragment ends provide evidence for a previously uncharacterized third step: the branched DNA strand is cleaved at one of several defined sites located within 15-16 nucleotides of the IES boundary, liberating the deleted DNA in a linear form.

Crystal structure of a Flp recombinase-Holliday junction complex: assembly of an active oligomer by helix swapping.

The crystal structure of a Flp recombinase tetramer bound to a Holliday junction intermediate has been determined at 2.65 A resolution. Only one of Flp's two domains, containing the active site, is structurally related to other lambda integrase family site-specific recombinases, such as Cre. The Flp active site differs, however, in that the helix containing the nucleophilic tyrosine is domain swapped, such that it cuts its DNA target in trans. The Flp tetramer displays pseudo four-fold symmetry matching that of the square planar Holliday junction substrate. This tetramer is stabilized by additional novel trans interactions among monomers. The structure illustrates how mechanistic unity is maintained on a chemical level while allowing for substantial variation on the structural level within a family of enzymes.

RecA protein promotes strand exchange with DNA substrates containing isoguanine and 5-methyl isocytosine.

The Escherichia coli RecA protein pairs homologous DNA molecules and promotes DNA strand exchange in vitro. We have examined DNA strand exchange between a 70 nucleotide ssDNA fragment and a 40 bp duplex, in which all G and C residues (at 18 positions distributed throughout the 40 bp exchanged region) were replaced with the nonstandard nucleosides 2'-deoxyisoguanosine (iG) and 2'-deoxy-5-methylisocytidine (MiC), respectively. We demonstrate that the nonstandard oligonucleotides are substrates for the RecA protein, permitting DNA strand exchange in vitro at a rate and efficiency comparable to exchange with normal DNA substrates. This observation provides an expanded experimental basis for discussions of potential roles for iG and MiC in a genetic code. Experiments of this type also provide another avenue for exploring RecA-facilitated DNA pairing mechanisms.

The importance of repairing stalled replication forks.

The bacterial SOS response to unusual levels of DNA damage has been recognized and studied for several decades. Pathways for re-establishing inactivated replication forks under normal growth conditions have received far less attention. In bacteria growing aerobically in the absence of SOS-inducing conditions, many replication forks encounter DNA damage, leading to inactivation. The pathways for fork reactivation involve the homologous recombination systems, are nonmutagenic, and integrate almost every aspect of DNA metabolism. On a frequency-of-use basis, these pathways represent the main function of bacterial DNA recombination systems, as well as the main function of a number of other enzymatic systems that are associated with replication and site-specific recombination.

Recombinational DNA repair in bacteria and the RecA protein.

In bacteria, the major function of homologous genetic recombination is recombinational DNA repair. This is not a process reserved only for rare double-strand breaks caused by ionizing radiation, nor is it limited to situations in which the SOS response has been induced. Recombinational DNA repair in bacteria is closely tied to the cellular replication systems, and it functions to repair damage at stalled replication forks, Studies with a variety of rec mutants, carried out under normal aerobic growth conditions, consistently suggest that at least 10-30% of all replication forks originating at the bacterial origin of replication are halted by DNA damage and must undergo recombinational DNA repair. The actual frequency may be much higher. Recombinational DNA repair is both the most complex and the least understood of bacterial DNA repair processes. When replication forks encounter a DNA lesion or strand break, repair is mediated by an adaptable set of pathways encompassing most of the enzymes involved in DNA metabolism. There are five separate enzymatic processes involved in these repair events: (1) The replication fork assembled at OriC stalls and/or collapses when encountering DNA damage. (2) Recombination enzymes provide a complementary strand for a lesion isolated in a single-strand gap, or reconstruct a branched DNA at the site of a double-strand break. (3) The phi X174-type primosome (or repair primosome) functions in the origin-independent reassembly of the replication fork. (4) The XerCD site-specific recombination system resolves the dimeric chromosomes that are the inevitable by-product of frequent recombination associated with recombinational DNA repair. (5) DNA excision repair and other repair systems eliminate lesions left behind in double-stranded DNA. The RecA protein plays a central role in the recombination phase of the process. Among its many activities, RecA protein is a motor protein, coupling the hydrolysis of ATP to the movement of DNA branches.

ATP hydrolysis and DNA binding by the Escherichia coli RecF protein.

The Escherichia coli RecF protein possesses a weak ATP hydrolytic activity. ATP hydrolysis leads to RecF dissociation from double-stranded (ds)DNA. The RecF protein is subject to precipitation and an accompanying inactivation in vitro when not bound to DNA. A mutant RecF protein that can bind but cannot hydrolyze ATP (RecF K36R) does not readily dissociate from dsDNA in the presence of ATP. This is in contrast to the limited dsDNA binding observed for wild-type RecF protein in the presence of ATP but is similar to dsDNA binding by wild-type RecF binding in the presence of the nonhydrolyzable ATP analog, adenosine 5'-O-(3-thio)triphosphate (ATPgammaS). In addition, wild-type RecF protein binds tightly to dsDNA in the presence of ATP at low pH where its ATPase activity is blocked. A transfer of RecF protein from labeled to unlabeled dsDNA is observed in the presence of ATP but not ATPgammaS. The transfer is slowed considerably when the RecR protein is also present. In competition experiments, RecF protein appears to bind at random locations on dsDNA and exhibits no special affinity for single strand/double strand junctions when bound to gapped DNA. Possible roles for the ATPase activity of RecF in the regulation of recombinational DNA repair are discussed.

Quantitative analysis of the kinetics of end-dependent disassembly of RecA filaments from ssDNA.

On linear single-stranded DNA, RecA filaments assemble and disassemble in the 5' to 3' direction. Monomers (or other units) associate at one end and dissociate from the other. ATP hydrolysis occurs throughout the filament. Dissociation can result when ATP is hydrolyzed by the monomer at the disassembly end. We have developed a comprehensive model for the end-dependent filament disassembly process. The model accounts not only for disassembly, but also for the limited reassembly that occurs as DNA is vacated by disassembling filaments. The overall process can be monitored quantitatively by following the resulting decline in DNA-dependent ATP hydrolysis. The rate of disassembly is highly pH dependent, being negligible at pH 6 and reaching a maximum at pH values above 7. 5. The rate of disassembly is not significantly affected by the concentration of free RecA protein within the experimental uncertainty. For filaments on single-stranded DNA, the monomer kcat for ATP hydrolysis is 30 min-1, and disassembly proceeds at a maximum rate of 60-70 monomers per minute per filament end. The latter rate is that predicted if the ATP hydrolytic cycles of adjacent monomers are not coupled in any way.

Cycling of inducibility of paroxysmal supraventricular tachycardia in women and its implications for timing of electrophysiologic procedures.

Arrhythmias in women may be affected by phases of the menstrual cycle. This study was designed to determine the prevalence of perimenstrual clustering of spontaneous episodes of paroxysmal supraventricular tachycardia (SVT) in women. It also tested the hypothesis that women with this temporal pattern of events have an altered probability of induction of paroxysmal SVT during electrophysiologic testing at higher estrogen states (midcycle or with estrogen replacement therapy) than at low estrogen states (perimenstrual or without estrogen replacement). A structured history of the relation of spontaneous paroxysmal SVTs to phases of the menstrual cycle was obtained prospectively among 42 women referred during a 3-year period. Patients with cyclical patterns of spontaneous tachycardias, who had had negative electrophysiologic studies at midcycle or while receiving estrogen replacement therapy, had repeat procedures (1) when premenstrual or at the onset of menses, or (2) after stopping estrogen replacement therapy. Seventeen of 42 consecutive female patients (40%) had histories of perimenstrual clustering of arrhythmias. Six women (4 with normal menstrual cycles, 2 on estrogen replacement therapy), who qualified for paired electrophysiologic studies because of a negative initial electrophysiologic study that included provocation with isoproterenol, had inducibility into SVTs during the second study. All 6 had dual atrioventricular (AV) nodal pathway physiology, 4 had AV nodal reentrant tachycardia (AVNRT) induced, 1 had both AVNRT and reciprocating AV tachycardias, and 1 had nonsustained AVNRT and an atrial tachycardia induced. Successful ablation procedures were performed in 5 of the 6 patients. Thus, among women with a history of perimenstrual clustering of paroxysmal SVT and among those receiving estrogen replacement therapy, scheduling of elective electrophysiologic procedures at times of low estrogen levels (premenstrual or off estrogen replacement therapy) may facilitate the probability of a successful procedure.

Sudden Wenckebach periods and their relationship to neurocardiogenic syncope.

Throughout a 9-month period during which 1,125 Holter tapes were reviewed prospectively we identified 13 nonmedicated patients with an arrhythmia, which for the purposes of this presentation was categorized, because of their mode of initiation, as sudden Wenckebach periods (WP). The episodes emerged abruptly from a normal (< or = 200 ms) PR interval with sudden prolongation of PR and PP intervals (and reversed PR-RP relationship) that took place over 1-8 cycles. The postpaced PR interval was shorter than that of the last conducted beat. The episodes were separated into two groups. Group I included 11 patients with symptoms other than syncope and Group II included 2 patients with syncope. There were 26 episodes of sudden WP in Group I. Twenty-five terminated in a single (and one in double) blocked P waves. Most episodes occurred between 10 PM and 7 AM. Symptoms did not correlate with the episodes. Mean 24-hour rates were < 90. In Group II there were 22 episodes, all occurring between 6 AM and 10 PM. The mean sinus cycle lengths before the phenomenon started to occur in Group I (861 +/- 185 ms) as well as the cycle lengths at the onset of block (1,096 +/- 215 ms) were statistically longer than those in Group II (591 +/- 40 ms and 747 +/- 63 ms, respectively, P < 0.0001). Although the mode of onset in the episodes in Group II was similar to Group I, 16 episodes terminated in 2-6 blocked P waves. Thus, the entire number of episodes could be categorized as an unusual type (because of the PR prolongation) of paroxysmal, or advanced second degree AV block. Because these patients had negative electrophysiological studies, positive tilt tests, and absent syncope after oral propranolol therapy, they were considered as having neurocardiogenic syncope. In addition, the faster than normal (> 100) mean 24-hour rates) suggested that they also had so-called inappropriate sinus tachycardia. In summary, Group I consisted of patients with a normal, benign, vagal-induced second-degree AV block, whereas the Holter findings in Group II appeared to reflect unusual (but natural, i.e., nonprovoked) electrocardiographic manifestations of certain patients with neurocardiogenic syncope.