Efficacious, nontoxigenic Bacillus anthracis spore vaccines based on strains expressing mutant variants of lethal toxin components.

We reported previously on the development of a Bacillus anthracis vaccine strain expressing high levels of recombinant protective antigen (rPA) [Cohen et al., Infec Immun 2000;68(8):4549-58]. To further explore the potential of the B. anthracis platform, we generated several attenuated strains expressing lethal toxin components PA and LF, which are biologically inactive, yet retain their antigenic properties. A single injection of 5 x 10(7) spores of one of these strains, carrying PA mutation at a site involved in effector translocation (residues 313-314) was shown to resemble wild type PA in inducing production of high levels of anti-PA neutralizing antibodies and producing effective protective immunity for 12 months. Long-term protection and persistence of functional antibody titers was observed after the gradual elimination of spores from guinea pig tissues 3 months after injection and in the measurable absence of bacteria in tissues. The mutant toxin components could, thus be an effective alternatives to their native counterparts when presented to the immune system in context of a live B. anthracis strain. These live vaccine prototypes may serve as a platform for future multi-component vaccines.

Use of a promoter trap system in Bacillus anthracis and Bacillus subtilis for the development of recombinant protective antigen-based vaccines.

We have recently reported Bacillus anthracis attenuated live vaccine strains efficiently expressing recombinant protective antigen (rPA) and have shown a direct correlation between the level of rPA secreted by these cells and efficacy (S. Cohen, I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak, M. Fisher, C. Kronman, B. Velan, and A. Shafferman, Infect. Immun. 68:4549-4558, 2000). To isolate more potent Bacillus promoters for a further increase in the production of rPA, we developed a promoter trap system based on various gfp reporter genes adapted for use in both Bacillus subtilis and B. anthracis backgrounds. Accordingly, a B. anthracis library of 6,000 clones harboring plasmids with chromosomal B. anthracis DNA fragments inserted upstream from gfpuv was constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent B. anthracis promoter identified (Pntr; 271 bp) was 500 times more potent than the native pagA promoter and 70 times more potent than the alpha-amylase promoter (Pamy). This very potent promoter was tested along with the other promoters (which are three, six, and eight times more potent than Pamy) for the ability to drive expression of rPA in either B. subtilis or B. anthracis. The number of cell-associated pre-PA molecules in B. anthracis was found to correlate well with the strength of the promoter. However, there appeared to be an upper limit to the amount of mature PA secreted into the medium, which did not exceed that driven by Pamy. Furthermore, the rPA constructs fused to the very potent promoters proved to be deleterious to the bacterial hosts and consequently led to genetic instability of the PA expression plasmid. Immunization with attenuated B. anthracis expressing rPA under the control of promoters more potent than Pamy was less efficient in eliciting anti-PA antibodies than that attained with Pamy. The results are consistent with the notion that overexpression of PA leads to severe secretion stress and have practical implications for the design of second-generation rPA-based vaccines.

In-vitro characterisation of the phagocytosis and fate of anthrax spores in macrophages and the effects of anti-PA antibody.

Antibodies (Abs) to the protective antigen (PA) component of the anthrax toxins have anti-spore as well as anti-toxin activities. Anti-PA antisera and purified anti-PA Abs enhance the phagocytosis by murine-derived macrophages (MQs) of spores of the Ames and Sterne strains and retard the germination of extracellular spores in vitro. The fate after phagocytosis of untreated and anti-PA-treated spores was further studied in culture medium that supported phagocytosis without stimulating spore germination (Dulbecco's minimal essential medium with horse serum 10%). The spores germinated within cells of primary peritoneal murine MQs (C3H/HeN) and MQs of the RAW264.7 MQ-like cell line; germination was associated with a rapid decline in spore viability. Exposure of MQs to inhibitors of phago-endosomal acidification (bafilomycin A and chloroquine) reduced the efficiency of MQ killing and allowed outgrowth and replication of the organisms. Treatment of spores with anti-PA Abs stimulated their phagocytosis and was associated with enhanced MQ killing of the spores. The enhanced killing of spores correlated with the greater extent of germination of anti-PA-treated spores after phagocytosis. A PA null mutant of the Ames strain exhibited none of the effects associated with anti-PA Ab treatment ofthe parental strain. Thus, the anti-PA Ab-specific immunity induced by vaccines has anti-spore activities and its role in impeding the early stages of infection with Bacillusanthracis needs to be assessed.

Attenuated nontoxinogenic and nonencapsulated recombinant Bacillus anthracis spore vaccines protect against anthrax.

Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 x 10(7) spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (>/=100 microgram/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.

Bovine acetylcholinesterase: cloning, expression and characterization.

The bovine acetylcholinesterase (BoAChE) gene was cloned from genomic DNA and its structure was determined. Five exons coding for the AChE T-subunit and the alternative H-subunit were identified and their organization suggests high conservation of structure in mammalian AChE genes. The deduced amino acid sequence of the bovine T-subunit is highly similar to the human sequence, showing differences at 34 positions only. However, the cloned BoAChE sequence differs from the published amino acid sequence of AChE isolated from fetal bovine serum (FBS) by: (1) 13 amino acids, 12 of which are conserved between BoAChE and human AChE, and (2) the presence of four rather than five potential N-glycosylation sites. The full coding sequence of the mature BoAChE T-subunit was expressed in human embryonal kidney 293 cells (HEK-293). The catalytic properties of recombinant BoAChE and its reactivity towards various inhibitors were similar to those of the native bovine enzyme. Soluble recombinant BoAChE is composed of monomers, dimers and tetramers, yet in contrast to FBS-AChE, tetramer formation is not efficient. Comparative SDS/PAGE analysis reveals that all four potential N-glycosylation sites identified by DNA sequencing appear to be utilized, and that recombinant BoAChE comigrates with FBS-AChE. A major difference between the recombinant enzyme and the native enzyme was observed when clearance from circulation was examined. The HEK-293-derived enzyme was cleared from the circulation at a much faster rate than FBS-AChE. This difference in behaviour, together with previous studies on the effect of post-translation modification on human AChE clearance [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959-967] suggests that cell-dependent glycosylation plays a key role in AChE circulatory residence.

Integration host factor binds to a unique class of complex repetitive extragenic DNA sequences in Escherichia coli.

Interspersed repeated DNA sequences are characteristic features of both prokaryotic and eukaryotic genomes. REP sequences are defined as conserved repetitive extragenic palindromic sequences and are found in Escherichia coli, Salmonella typhimurium and other closely related enteric bacteria. These REP sequences may participate in the folding of the bacterial chromosome. In this work we describe a unique class of 28 conserved complex REP clusters, about 100bp long, in which two inverted REPs are separated by a singular integration host factor (IHF) recognition sequence. We term these sequences RIP (for repetitive IHF-binding palindromic) elements and demonstrate that IHF binds to them specifically. It is estimated that there are about 70 RIP elements in E. coli. Our analysis shows that the RIP elements are evenly distributed around the bacterial chromosome. The possible function of the RIP element is discussed.

Genetic and biochemical analysis of the integration host factor of Escherichia coli.

Integration host factor (IHF) is a small, heterodimeric DNA-binding protein of Escherichia coli composed of two subunits, alpha and beta, encoded by the himA and hip genes, respectively. IHF binds to the minor groove at a consensus sequence and bends DNA. We mutagenized the hip gene and studied the activity of the mutant IHF proteins in vivo and in vitro. Substitutions at the C-terminal alpha-helix (alpha-helix 3) reduced IHF activity and relaxed the specificity to DNA without abolishing the ability of IHF to bend DNA. These results indicate that the C-terminal region of Hip participates in determining IHF specificity. Alanine substitutions in beta-strands 2 and 3 generally had no effect on IHF activity in vivo suggesting that individually, many of these residues make only small contributions to the binding of IHF to DNA. Replacing the single amino acid of Hip that differs from HU in a highly conserved region of the arm did not affect IHF activity. This finding led us to conclude that this region of Hip does not contribute to specific DNA recognition by IHF. The binding of IHF to DNA is probably not restricted to one domain, but requires the co-operative participation of a number of regions of the protein.

Genes coding for integration host factor are conserved in gram-negative bacteria.

A genetic system for the selection of clones coding for integration host factor and HU homologs is described. We demonstrate that the himA and hip genes of Serratia marcescens and Aeromonas proteolytica can substitute for the Escherichia coli genes in a variety of biological assays. We find that the sequence and genetic organization of the himA and hip genes of S. marcescens are highly conserved.

HU and integration host factor function as auxiliary proteins in cleavage of phage lambda cohesive ends by terminase.

HU and integration host factor (IHF) are small, basic heterodimeric DNA-binding proteins which participate in transcription initiation, DNA replication, and recombination. We constructed isogenic Escherichia coli strains in which HU, IHF, or both proteins were absent. Bacteriophage lambda did not grow in hosts lacking both HU and IHF. Phage DNA replication and late gene transcription were normal in the double mutants, but packaging of lambda DNA was defective. Mature phage DNA molecules were absent, indicating that terminase was unable to linearize lambda DNA. Phage variants carrying a small substitution near cos or the ohm1 mutation in the terminase gene, Nul, formed plaques on HU- IHF- strains. We propose that HU or IHF is required to establish the higher-order DNA-protein structure at cos that is the substrate for lambda terminase.

Ornithine decarboxylase induction in psoralen-treated mouse epidermis used as a test of UV-A sunscreen potency.

Inhibition by sunscreens of UV-A-induced epidermal ornithine decarboxylase (ODC) activity in 8-methoxypsoralen-treated mice was used to examine the UV-A sunscreen activity of two commercial preparations, Uval (para-aminobenzoic acid) and PreSun (a benzophenone derivative). Both gave significant protection against UV-A doses of up to 3 J/cm2 and Uval was more effective that PreSun in the UV-A range. At intermediate doses (5-15 J/cm2) neither Uval nor PreSun significantly altered the ODC response to UV-A. At doses of 20-40 J/cm2 UV-A, control and PreSun-treated animals showed a decline in ODC activity which histological studies suggested was due to severe epidermal damage. This decline in ODC activity and severe epidermal damage was not seen in Uval-treated animals. Topical zinc oxide was highly effective in preventing induction of ODC by UV-A irradiation at doses up to 40 J/cm2, and was significantly more effective than either PreSun or Uval.

Neonatal 5-oxoprolinuria: difficult-to-diagnose?

A male newborn infant presented with metabolic acidosis and haemolytic anaemia. Renal tubular acidosis was suspected in the absence of amino aciduria and the patient was treated with sodium bicarbonate. Two years later, the chronic acidosis, clinical observation of developmental delay and ataxia prompted further investigational studies. 5-Oxoprolinuria was identified by gas-liquid chromatography and confirmed by mass spectrometry after an initial mass spectrum analysis reported a glutamic acid artifact. Glutathione and glutathione synthetase in erythrocytes were 25% and 5% of control values, respectively. On the basis of neonatal metabolic acidosis, without amino aciduria and an elevated reticulocyte count, a recommendation is made for blood glutathione and urine 5-oxoproline screening, followed by glutathione synthetase assay for confirmation of neonatal 5-oxoprolinuria.

Psoralen-containing sunscreen induces phototoxicity and epidermal ornithine decarboxylase activity.

Sunscreens containing 5-methoxypsoralen (5-MOP) are being promoted commercially to increase suntanning and sun protection. A recent study indicated that the 5-MOP concentration used in these sunscreens is too low to induce cutaneous phototoxicity with ultraviolet (UV) radiation. We investigated whether the sunscreen Sun System III (SS III), which contains 5-MOP, could induce skin erythema, edema, delayed pigmentation, and epidermal ornithine decarboxylase (ODC) activity when used in conjunction with UVA radiation (320-400 nm). ODC induction is an early event in the promotion of skin tumors. Increased epidermal ODC activity has been reported after exposure to UVB radiation (290-320 nm) alone and with topical 8-methoxypsoralen (8-MOP) plus UVA radiation. Using a solar simulator, we found SS III-induced erythema, edema, and epidermal ODC activity in hairless mouse skin with only 5 joules/cm2 of UVA. Human skin showed erythema and delayed pigmentation with SS III plus 20 joules/cm2 of UVA. No phototoxicity was seen in human skin unless the solar simulator output was filtered through water to reduce infrared radiation. This indicates that cutaneous phototoxic reactions to 5-MOP plus UVA are diminished by heat. Like 8-MOP, 5-MOP cross-links DNA and has the same skin photocarcinogenic potential as 8-MOP. Therefore the use of phototoxic psoralens in over-the-counter sunscreens is inappropriate because of the risk of increased UV-induced skin cancer.

A strategy for glycine encephalopathy therapy.

An inherited defect in the glycine cleavage enzyme results in the condition of neonatal glycine encephalopathy which has not responded to the current innovative methods of therapy. A re-examination of the enzyme structure and metabolic pathways, leads us to recommend future clinical evaluation of (1) vitamin-responsiveness, e.g., pyridoxine, folate and lipoic acid, (2) methionine, (3) N5, N10-methylene tetrahydrofolate and (4) alpha-methylserine therapy during the critical period of neonatal brain growth and development.

Photoaugmentation in the hairless mouse: a study using ornithine decarboxylase activity and alteration of DNA synthesis as markers of epidermal response.

Photoaugmentation is the potentiation of UVB-induced cutaneous erythema by UV irradiation. We have examined other cutaneous responses to UVB irradiation-the 4 hr depression of DNA synthesis, the 48 hr stimulation of DNA synthesis, and the induction of ornithine decarboxylase (ODC), to determine whether these were also susceptible to augmentation by UVA, which does not cause these responses when administered alone. No photoaugmentation of DNA synthesis, stimulation or ODC induction occurred. The early depression of DNA synthesis was slightly augmented for this did not consistently reach significance.

Biochemical and clinical studies of a new case of alpha-aminoadipic aciduria.

A mentally retarded, 10-year-old female with obesity, hypotonia, clumsiness and mild ocular abnormalities excreted in her urine large amounts of alpha-aminoadipic acid. Amino acid analyser studies and gas-liquid chromatography--mass spectrometry (GC--MS) confirmed the presence of alpha-aminoadipic acid in both urine and plasma but, in contrast to most other patients with this disorder, failed to demonstrate significant levels of alpha-ketoadipic acid in urine. Other known causes of alpha-aminoadipic aciduria were eliminated by showing that levels of lysine, saccharopine and pipecolic acid in plasma and urine were normal and that the activity of glutaryl-CoA dehydrogenase was also normal. Loading with L-lysine and L-tryptophan both increased the concentration of alpha-aminoadipic acid in blood and urine compatible with the primary deficiency of alpha-ketoadipate dehydrogenase, in spite of the absence of alpha-ketoadipic aciduria. Dietary restriction of lysine and administration of vitamins B1 and B6 were unsuccessful in correcting the biochemical abnormality.

Peptides readily penetrate the blood-brain barrier: uptake of peptides by synaptosomes is passive.

The intracarotid artery quick injection technique of Oldendorf was utilized to determine the Brain Uptake Index (BUI) of radio-labeled peptides in comparison with 3H2O or 14C-antipyrine as counterlabels. The normalized BUI values for 3H-MIF-I, 3H-alpha-MSH and 14C-AVP were 13.7, 9.6 and 13.0 respectively at 15 sec after injection consistent with their having readily penetrated the blood-brain barrier. The BUI values were similar, though somewhat increased, at 10 min postinjection consistent with their ready exit across the blood-brain barrier. At 15 sec after injection 0.5+/-0.1%/g brain of the originally injected peptide label was recovered; and 0.1+/-0.2%/g brain was recovered after 10 min. The label was distributed uniformly in the major brain regions at both times. However, the percentage of the originally injected label/g of pineal and pituitary gland tissue was 10-20 X increased as compared with the major brain regions as would be expected by their location outside the blood-brain barrier. The in vitro uptake of the radio-labeled peptides by synaptosomes prepared from the whole brain and the major brain regions was passive; it was not temperature dependent, nor was it Na+ dependent. However, the binding of the three peptides by the synaptosomes varied considerably: AVP greater than MSH greater MIF: 50 greater than 5 greater 1. The penetratin of the blood-brain barrier by the three peptides is consistent with their having CNS effects.

Synthesis of viral proteins by the avian myeloblastosis viral core component.

Avian Myeloblastosis Viral (AMV) core component was isolated and shown to synthesize AMV proteins in vitro. This reaction was linearly dependent on viral core concentration, proceeded linearly with time, and was inhibited by puromycin and aurintricarboxylic acid. The proteins synthesized in vitro co-electrophoresed and co-chromatographed with known proteins, and were immunoprecipitated by total and monospecific antibodies to known AMV proteins.

Relationship between RNA polymerase and protein kinase activities in rat mammary gland nuclei.

1. Extracts from rat mammary gland nuclei contain cyclic AMP -independent protein kinases which phosphorylate casein rather than histone. 2. A major increase in nuclear protein kinase activity occurred during late pregnancy and was maintained with the onset of lactation. 3. Two major peaks of activity were resolved by chomatography of nuclear extracts on DEAE-Sephadex; the first (NI) appeared in the void volume and the second (NII) was eluted by 0.05-0.12 M ammonium sulfate. Several other regions of lesser activity were also present. 4. Protein kinases in the cytosol 105,000 times g supernatant, precipitated by 70 percent ammonium sulfate, dialyzed against buffer, and chromatographed on DEAE-Sephadex, yielded a major components phosphorylated histone in preference to casein, and this was stimulated by cyclic AMP if histone was the substrate, but only the first (void volume) fraction was cyclic AMP-dependent when casein was used. 5. Most of RNA polymerases Ib and II, derived from the nucleolus and nucleoplasm, respectively, appeared in column fractions distinct from those containing the major NI and NII protein kinases. 6. Cyclic AMP altered the amount of RNA product synthesized by polymerases Ib and II, but the explanation for this is unknown. Due to their elution profiles and cyclic AMP-independence, protein kinases NI and NII are excluded from playing a catalytic role in these effects; participation of quantitatively minor protein kinases which co-elute with polymerase Ib and II is not yet excluded.