Neutralizing chimeric mouse-human antibodies against Burkholderia pseudomallei protease: expression, purification and characterization

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2 % glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.     

A randomised, double-blind, controlled vaccine efficacy trial of DNA/MVA ME-TRAP against malaria infection in Gambian adults

Background

Many malaria vaccines are currently in development, although very few have been evaluated for efficacy in the field. Plasmodium falciparum multiple epitope (ME)– thrombospondin-related adhesion protein (TRAP) candidate vaccines are designed to potently induce effector T cells and so are a departure from earlier malaria vaccines evaluated in the field in terms of their mechanism of action. ME-TRAP vaccines encode a polyepitope string and the TRAP sporozoite antigen. Two vaccine vectors encoding ME-TRAP, plasmid DNA and modified vaccinia virus Ankara (MVA), when used sequentially in a prime-boost immunisation regime, induce high frequencies of effector T cells and partial protection, manifest as delay in time to parasitaemia, in a clinical challenge model.

Methods and Findings

A total of 372 Gambian men aged 15–45 y were randomised to receive either DNA ME-TRAP followed by MVA ME-TRAP or rabies vaccine (control). Of these men, 296 received three doses of vaccine timed to coincide with the beginning of the transmission season (141 in the DNA/MVA group and 155 in the rabies group) and were followed up. Volunteers were given sulphadoxine/pyrimethamine 2 wk before the final vaccination. Blood smears were collected weekly for 11 wk and whenever a volunteer developed symptoms compatible with malaria during the transmission season. The primary endpoint was time to first infection with asexual P. falciparum. Analysis was per protocol.DNA ME-TRAP and MVA ME-TRAP were safe and well-tolerated. Effector T cell responses to a non-vaccine strain of TRAP were 50-fold higher postvaccination in the malaria vaccine group than in the rabies vaccine group. Vaccine efficacy, adjusted for confounding factors, was 10.3% (95% confidence interval, −22% to +34%; p = 0.49). Incidence of malaria infection decreased with increasing age and was associated with ethnicity.

Conclusions

DNA/MVA heterologous prime-boost vaccination is safe and highly immunogenic for effector T cell induction in a malaria-endemic area. But despite having produced a substantial reduction in liver-stage parasites in challenge studies of non-immune volunteers, this first generation T cell–inducing vaccine was ineffective at reducing the natural infection rate in semi-immune African adults.     

Brain choline concentrations may not be altered in euthymic bipolar disorder patients chronically treated with either lithium or sodium valproate

BACKGROUND: It has been suggested that lithium increases choline concentrations, although previous human studies examining this possibility using 1H magnetic resonance spectroscopy (1H MRS) have had mixed results: some found increases while most found no differences. METHODS: The present study utilized 1H MRS, in a 3 T scanner to examine the effects of both lithium and sodium valproate upon choline concentrations in treated euthymic bipolar patients utilizing two different methodologies. In the first part of the study healthy controls (n = 18) were compared with euthymic Bipolar Disorder patients (Type I and Type II) who were taking either lithium (n = 14) or sodium valproate (n = 11), and temporal lobe choline/creatine (Cho/Cr) ratios were determined. In the second part we examined a separate group of euthymic Bipolar Disorder Type I patients taking sodium valproate (n = 9) and compared these to controls (n = 11). Here we measured the absolute concentrations of choline in both temporal and frontal lobes. RESULTS: The results from the first part of the study showed that bipolar patients chronically treated with both lithium and sodium valproate had significantly reduced temporal lobe Cho/Cr ratios. In contrast, in the second part of the study, there were no effects of sodium valproate on either absolute choline concentrations or on Cho/Cr ratios in either temporal or frontal lobes. CONCLUSIONS: These findings suggest that measuring Cho/Cr ratios may not accurately reflect brain choline concentrations. In addition, the results do not support previous suggestions that either lithium or valproate increases choline concentrations in bipolar patients.     

Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans

Protective immunity against Mycobacterium tuberculosis depends on the generation of a T(H)1-type cellular immune response, characterized by the secretion of interferon-gamma (IFN-gamma) from antigen-specific T cells. The induction of potent cellular immune responses by vaccination in humans has proven difficult. Recombinant viral vectors, especially poxviruses and adenoviruses, are particularly effective at boosting previously primed CD4(+) and CD8(+) T-cell responses against a number of intracellular pathogens in animal studies. In the first phase 1 study of any candidate subunit vaccine against tuberculosis, recombinant modified vaccinia virus Ankara (MVA) expressing antigen 85A (MVA85A) was found to induce high levels of antigen-specific IFN-gamma-secreting T cells when used alone in bacille Calmette-Guerin (BCG)-naive healthy volunteers. In volunteers who had been vaccinated 0.5-38 years previously with BCG, substantially higher levels of antigen-specific IFN-gamma-secreting T cells were induced, and at 24 weeks after vaccination these levels were 5-30 times greater than in vaccinees administered a single BCG vaccination. Boosting vaccinations with MVA85A could offer a practical and efficient strategy for enhancing and prolonging antimycobacterial immunity in tuberculosis-endemic areas.     

Potassium channel activators based on the benzopyran substructure: synthesis and activity of the C-8 substituent

The synthesis of a series of methoxy bearing 2,2-dimethyl-2H-1-benzopyrans have been achieved for testing as potassium channel activators. The synthesis involves formation of 6-cyano-8-methoxy-2,2-dimethyl-2H-1-benzopyran from vanillin, epoxidation, then ring opening of the epoxide with nitrogen nucleophiles to produce the new benzopyrans. Biological testing showed a dramatic decrease in activity thus revealing an important site of activity in this class of compounds.     

A live attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses

Previous evaluations of inactivated whole-virus and envelope subunit vaccines to equine infectious anemia virus (EIAV) have revealed a broad spectrum of efficacy ranging from highly type-specific protection to severe enhancement of viral replication and disease in experimentally immunized equids. Among experimental animal lentivirus vaccines, immunizations with live attenuated viral strains have proven most effective, but the vaccine efficacy has been shown to be highly dependent on the nature and severity of the vaccine virus attenuation. We describe here for the first time the characterization of an experimental attenuated proviral vaccine, EIAV(UK)deltaS2, based on inactivation of the S2 accessory gene to down regulate in vivo replication without affecting in vitro growth properties. The results of these studies demonstrated that immunization with EIAV(UK)deltaS2 elicited mature virus-specific immune responses by 6 months and that this vaccine immunity provided protection from disease and detectable infection by intravenous challenge with a reference virulent biological clone, EIAV(PV). This level of protection was observed in each of the six experimental horses challenged with the reference virulent EIAV(PV) by using a low-dose multiple-exposure protocol (three administrations of 10 median horse infectious doses [HID(50)], intravenous) designed to mimic field exposures and in all three experimentally immunized ponies challenged intravenously with a single inoculation of 3,000 HID(50). In contrast, na?ve equids subjected to the low- or high-dose challenge develop a detectable infection of challenge virus and acute disease within several weeks. Thus, these data demonstrate that the EIAV S2 gene provides an optimal site for modification to achieve the necessary balance between attenuation to suppress virulence and replication potential to sufficiently drive host immune responses to produce vaccine immunity to viral exposure.     

Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons

Loss-of-function mutations in parkin are the major cause of early-onset familial Parkinson's disease. To investigate the pathogenic mechanism by which loss of parkin function causes Parkinson's disease, we generated a mouse model bearing a germline disruption in parkin. Parkin-/- mice are viable and exhibit grossly normal brain morphology. Quantitative in vivo microdialysis revealed an increase in extracellular dopamine concentration in the striatum of parkin-/- mice. Intracellular recordings of medium-sized striatal spiny neurons showed that greater currents are required to induce synaptic responses, suggesting a reduction in synaptic excitability in the absence of parkin. Furthermore, parkin-/- mice exhibit deficits in behavioral paradigms sensitive to dysfunction of the nigrostriatal pathway. The number of dopaminergic neurons in the substantia nigra of parkin-/- mice, however, is normal up to the age of 24 months, in contrast to the substantial loss of nigral neurons characteristic of Parkinson's disease. Steady-state levels of CDCrel-1, synphilin-1, and alpha-synuclein, which were identified previously as substrates of the E3 ubiquitin ligase activity of parkin, are unaltered in parkin-/- brains. Together these findings provide the first evidence for a novel role of parkin in dopamine regulation and nigrostriatal function, and a non-essential role of parkin in the survival of nigral neurons in mice.     

Mental retardation and abnormal skeletal development (Dyggve-Melchior-Clausen dysplasia) due to mutations in a novel,evolutionarily conserved gene

Dyggve-Melchior-Clausen dysplasia (DMC) and Smith-McCort dysplasia (SMC) are similar, rare autosomal recessive osteochondrodysplasias. The radiographic features and cartilage histology in DMC and SMC are identical. However, patients with DMC exhibit significant developmental delay and mental retardation, the major features that distinguish the two conditions. Linkage studies localized the SMC and DMC disease genes to chromosome 18q12-21.1, providing evidence suggesting that they are allelic disorders. Sequence analysis of the coding exons of the FLJ90130 gene, a highly evolutionarily conserved gene within the recombination interval defined in the linkage study, identified mutations in SMC and DMC patients. The affected individuals in two consanguinous DMC families were homozygous for a stop codon mutation and a frameshift mutation, respectively, demonstrating that DMC represents the FLJ90130-null phenotype. The data confirm the hypothesis that SMC and DMC are allelic disorders and identify a gene necessary for normal skeletal development and brain function.