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The etiological agent of anthrax disease in animals and humans is the spore-forming bacterium Bacillus anthracis. The major factors of virulence of B. anthracis are located on two plasmids, pXO1 and pXO2. pXO2 encodes a poly-d-glutamic acid capsule (19, 41), while pXO1 encodes two binary exotoxins, the lethal toxin (LT) and the edema toxin (ET) (43, 46, 61). These two toxins are composed of three different proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF) (for a review, see reference 36). PA is the common receptor binding domain of the toxins and can interact with the two different effector domains, EF and LF, to mediate their entry into target cells (14). EF is a calmodulin-dependent adenylate cyclase (37) responsible for the edema seen at the site of infection in experimental animals (17). The LF is a metalloprotease (34) recently shown to cleave the amino termini of the mitogen-activated protein kinase kinases 1 and 2, which results in their inactivation (13). It remains to be determined whether these are the main physiological substrates for the LT activity in vivo (5, 22). Two types of anthrax vaccines are licensed for use in humans: the spores of the toxigenic, nonencapsulated B. anthracis STI-1 strain (55) and the cell-free PA-based vaccines consisting of aluminum hydroxide-adsorbed supernatant material from cultures of the toxigenic, nonencapsulated B. anthracis strain V770-NPI-R (49) or alum-precipitated culture filtrate from the Sterne strain (6). The use of the live attenuated STI-1 occasionally results in general and local adverse responses, observed both after primary application and revaccination, and the frequency of responses increases with the number of vaccinations (58). Furthermore, it was reported that the STI-1 vaccine has a relatively low immunogenicity (reviewed by Stepanov et al. in reference 58). To increase the immunogenicity, a combined vaccine of live STI-1 supplemented with cell-free PA formulation was evaluated and proposed for veterinary use (1). While the cell-free PA-based vaccines appear to be safer, they require numerous boosters (8) and were shown to have reduced ability to protect laboratory animals against certain virulent strains of B. anthracis (39, 60). In addition, these vaccines contain variable amounts of PA, as well as undefined quantities of LF and EF, adsorbed to aluminum hydroxide (4, 21, 49, 59). It appears, therefore, that there is a need for a safe and more efficient vaccine which could generate stable and prolonged immunity in humans (59). These conclusions led to the evaluation of various adjuvants with purified PA (2, 16, 29, 59) and to the creation of two types of live vaccines: vaccines based on nonvirulent B. anthracis (pXO1+) mutated strains (31, 47) and vaccines expressing PA from a cloned pagA gene using heterologous hosts such as the vaccinia virus, Bacillus subtilis, Salmonella typhimurium (10, 27, 28, 30, 31, 64), or a nontoxinogenic strain of B. anthracis (4). These pioneering studies suggest that recombinant B. anthracis live vaccines may have potential as a future anthrax vaccine. We report here the construction of several recombinant, nonencapsulated, and nontoxinogenic B. anthracis spore-forming strains expressing different levels of PA. We demonstrate that one of these strains, containing the pagA gene under a potent heterologous constitutive promoter, can be safely used to provide efficacious long-lasting immunity in experimental animals following a single immunization dose.

Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.

In an attempt to identify the target recognition molecule(s) involved in the interaction between CD3− large granular lymphocyte (LGL) and a tumor cell target, monoclonal antibodies (mAb) to NK-susceptible K562 tumor cell membrane glycoproteins were developed. After screening by ELISA for reactivity to K562 membrane glycoproteins, two monoclonal antibodies were identified (mAb 35 and mAb 36). One of the monoclonal antibodies (mAb 36) was found to inhibit conjugation between LGL and K562 target cells and also to inhibit lysis of K562 by LGL. Upon further testing, mAb 36 also inhibited the binding between LGL and other NK-susceptible target cells, e.g., Daudi and Molt 4. In contrast, mAb 35, even though binding to K562, did not inhibit the binding of LGL to tumor targets and therefore was used as an isotype control. When mAb 36 was utilized as an affinity matrix, bound proteins specifically inhibited CD3− LGL-K562 conjugation. Experiments involving tunicamycin treatment of tumor target cells demonstrated that mAb 36 recognized a carbohydrate moiety rather than the protein core. Therefore, these data suggested that the target cell recognition molecule which is recognized by mAb 36 appears to be a membrane carbohydrate-associated molecule.

In a search for novel attenuated vaccine candidates for use againstYersinia pestis, the causative agent of plague, a signature-tagged mutagenesis strategy was used and optimized for a subcutaneously infected mouse model. A library of tagged mutants of the virulentY. pestisKimberley53 strain was generated. Screening of 300 mutants through two consecutive cycles resulted in selection of 16 mutant strains that were undetectable in spleens 48 h postinfection. Each of these mutants was evaluated in vivo by assays for competition against the wild-type strain and for virulence following inoculation of 100 CFU (equivalent to 100 50% lethal doses [LD50] of the wild type). A wide spectrum of attenuation was obtained, ranging from avirulent mutants exhibiting competition indices of 10−5to 10−7to virulent mutants exhibiting a delay in the mean time to death or mutants indistinguishable from the wild type in the two assays. Characterization of the phenotypes and genotypes of the selected mutants led to identification of virulence-associated genes coding for factors involved in global bacterial physiology (e.g.,purH,purK,dnaE, andgreA) or for hypothetical polypeptides, as well as for the virulence regulator genelcrF. One of the avirulent mutant strains (LD50, >107CFU) was found to be disrupted in thepcmlocus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcmmutant was superior to the EV76 live vaccine strain because it induced 10- to 100-fold-higher antibody titers to the protective V and F1 antigens and because it conferred efficacious protective immunity.

The fraction 1 capsular protein (F1) is considered an important but not essential virulence factor unique to Y. pestis (Welkos et al., 1995). Immunization with the F1 protein has been shown to protect mice against subcutaneous challenge with wild type Y. pestis (Andrews et al., 1996) and a combined formulation containing F1 and V antigen confers protection against airborne infection (Williamson et al., 1997). The protein has been associated with eliciting protective immune response in humans as well. The observation that genetic immunization is able to elicit a protective immunity has fostered a new generation in vaccine development. The caf1 gene, which codes for the F1 protein, was previously used as DNA vaccine. In this study inbred mice were found to be non responsive, and outbred mice responded by a weak anamnestic response (Brandler et al., 1998). The advent in genetic vaccination and the accumulating information on factors modulating the extent of response to DNA vaccines led us to re-examine genetic vaccination based on F1 antigen. Here we compare three F1 DNA derivatives carrying different signals for cellular localization and demonstrate that one such genetic derivative, which presumably targets expression to the cytosol induces an effective antibody response and confers protection against high doses of infective Y. pestis.

The role of the functional architecture of the human acetylcholinesterase (HuAChE) active centre in accommodating the non-covalent inhibitors tacrine and huperzine A, or the carbamates pyridostigmine and physostigmine, was analysed using 16 mutants of residues lining the active-centre gorge. Despite the structural diversity of the ligands, certain common properties of the complexes could be observed: (a) replacement of aromatic residues Tyr133, Tyr337 and especially Trp86, resulted in pronounced changes in stability of all the complexes examined; (b) effects due to replacements of the five other aromatic residues along the active-centre gorge, such as the acyl pocket (Phe295, Phe297) or at the peripheral anionic site (Tyr124, Trp286, Tyr341) were relatively small; (c) effects due to substitution of the carboxylic residues in the gorge (Glu202, Glu450) were moderate. These results and molecular modelling indicate that the aromatic side chains of residues Trp86, Tyr133 and Tyr337 form together a continuous ‘aromatic patch ’ lining the wall of the active-centre gorge, allowing for the accommodation of the different ligands via multiple modes of interaction. Studies with HuAChE mutants carrying replacements at positions 86, 133 and 337 indicate that the orientations of huperzine A and tacrine in the HuAChE complexes in solution are significantly different from those observed in X-ray structures of the corresponding complexes with Torpedo californica AChE (TcAChE). These discrepancies may be explained in terms of structural differences between the complexes of HuAChE and TcAChE or, more likely, by the enhanced flexibility of the AChE active-centre gorge in solution as compared with the crystalline state.

Interactions of the potential therapeutic agents tacrine, huperzine A and edrophonium, with 15 mutants of human acetylcholinesterase (HuAChE) were investigated. The score of new inhibition and dissociation constants measured, expand considerably the corresponding data found in literature. Despite the structural diversity of the ligands certain common properties of the complexes could be observed: a. replacement of aromatic residues Y133, Y337 and especially W86, resulted in pronounced changes in stability of all the complexes examined, b. effects due to replacements of the five other aromatic residues along the active-center gorge such as the acyl pocket (F295, F297) or at the peripheral anionic site (Y124, W286, Y341) were relatively small, c. effects due to substitution of the carboxylic residues in the gorge (E202, E450) were moderate. These results and the corresponding molecular models indicate that the aromatic side chains of residues W86, Y133 and Y337 form together a continuous “aromatic patch” lining the wall of the active center gorge allowing for the accommodation of the different ligands via various modes of interactions (cation-π, π-π, H-bond, hydrophobic). The HuAChE-bound orientations of edrophonium, huperzine A and tacrine inferred from the mutagenesis studies, were compared to those in the available X-ray structures of the corresponding complexes with Torpedo californica AChE (TcAChE). For edrophonium the two structures are very similar with respect to the ligand and its binding environment. On the other hand, the x-ray orientation for tacrine and huperzine A is inconsistent with mutagenesis results - some mutations do not support expected interactions while others suggest interactions that are not observed in the x-ray structures. These inconsistencies may be explained in terms of: a. structural differences between the complexes of HuAChE and TcAChE, even though the active center regions of the two enzymes, consisting of over 30 residues, differ in only one position [Y337(F330)]; b. the active-center gorge of AChE in solution is more flexible than in the crystalline state allowing for modified binding orientations of the same ligand in the two states.

Senile dementia of the Alzheimer’s type (SDAT) is characterised by loss of cholinergic neuronal markers like the enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), in selected brain regions (Bierer et al., 1995). These indications of progressive depletion of cholinergic synapses led to the hypothesis that increasing the central nervous system (CNS) levels of acetylcholine (ACh), through inhibition of AChE, will improve cognition in SDAT (Court & Perry, 1991). This approach is theoretically preferable to other means of cholinergic augmentation since it may amplify the natural temporal pattern of ACh release, rather than globally stimulating the cholinergic system. Yet up to the present most of these agents show only mild to moderate ameliorating effects on memory deficits (Schneider & Tariot, 1994; Kan, 1992). To further optimize the therapeutic efficacy of these agents, a better understanding is needed of the structural features determining their interactions with AChE. The recent progress in the elucidation of structure-function characteristics of AChE, is therefore of considerable importance for these efforts.

The surface Ω loop (Cys69-Cys96) in human acetylcholinesterase (HuAChE) was recently implicated by molecular simulations in enhancing substrate and ligand accessibility to the active center and in the allosteric modulation of enzymatic activity. In lipases which together with AChEs belong to the esterase/lipase family sharing the α/β hydrolase fold, such Ω loop mobility was also shown to be functionally significant. In order to examine the relation between the loop structure and reactivity characteristics of the enzyme, several mutant HuAChE’s carrying modifications of the loop sequence were generated and kinetically evaluated. These modifications included replacements at the loop ends (Tyr72, Glu95), substitution of residues involved in hydrogen-bond interactions of the loop with the protein core (Tyr77, Glu84) as well as residues participating in such interactions within the loop (Met85, Asn87), replacement of the conserved proline residues (Pro 78, Pro 88) and deletion of 5 residue segment (Pro 78-Gly82) from the loop sequence. Most of the residue replacements, including those of the prolines, had very limited or no effect on enzyme reactivity. These findings suggest that unlike the case of lipase the Ω loop in HuAChE is not involved in in large lid-like motions. In cases where modifications of loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp86 as implied by the altered catalytic activity, the interactions with quaternary ligands and the effect on the inhibitory activity of the peripheral site ligand propidium.

Many members of the alphavirus family are important human or veterinary pathogens. These viruses are extremely similar in molecular architecture yet differ in host range and in the pathological consequences of infection. Despite marked structural conservation, the various species are inefficient in eliciting immunological cross protection. Previously(l) we described a systematic approach for selection of epitope-cassettes of the E2 envelope of the alphavirus family that induce virus species specific protective immunity. This approach was tested and proved successful(2–5) with two viruses - Sindbis (SIN) and Semliki Forest (SF) - which are very remote phylogenetically. Vaccination was performed with recombinant peptide cassettes fused to a bacterial protein carrier (β-galactosidase) formulated with different adjuvants. In order to create a vaccine compatible with human use, we designed several live vector vaccines for presentation of the protective epitope cassette. Attenuated salmonella vaccine vector which can potentially allow presentation of many different cassettes and thus generation of a pan-alphavirus vaccine, proved to be inefficient. On the other hand the live attenuated viral vaccine based on a benign alphavirus such as SIN vector, appears to be promising. The principle of such vaccines is substitution of E2 epitopes of SIN virus vector with parallel protective epitopes from a different alphavirus. This system should have the advantage of presentation of specific virus epitopes in the context of the SIN alphavirus backbone which in turn could provide cross protective immunity on the basis of multiple common antigen determinants.

Acetylcholinesterase is a serine hydrolase whose function at the cholinergic synapse, is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh). The recently resolved 3D structure of Torpedo californica AChE (TcAChE) revealed a deep and narrow ‘gorge’, which penetrates halfway into the enzyme and contains the catalytic site at about 4A from its base (Sussman et al., 1991). The active center interacts with ACh through several subsites including the catalytic triad (Ser203(200), His447(440), Glu334(327): Sussman et al., 1991; Gibney et al., 1990; Shafferman et al., 1992a, b), the oxyanion hole (Gly121(119), Gly 122(120), Ala204(201); Sussman et al., 1991), the acyl pocket (Phe295 (288) and Phe297(290); Vellom et al., 1993; Ordentlich et al., 1993a).

The metabolic fate of human leukocyte interferon (HuIFN-alpha) was studied after intravenous injection into rats and cynomolgus monkeys. At various intervals the animals were sacrificed and the HuIFN-alpha content determined in serum and various tissues. HuIFN-alpha quickly disappeared from the circulation and was found mainly in the kidneys, in which levels were at least 7- to 10-fold higher than in the liver, spleen, lungs, heart, brain and muscles. No interferon was detected in urine. Subcellular fractionation of kidney revealed that the mitochondrial-lysosomal fraction (15 000 g) had a high HuIFN-alpha content. It was also found that HuIFN-alpha was rapidly inactivated by two types of proteinases found in the lysosomal fractions of rat, monkey and human kidneys, with an optimal pH of 3 to 4. The inactivation was partially inhibited by either pepstatin or leupeptin. Inactivation was totally prevented by a mixture of both inhibitors. Since it is known that interferon is scantily excreted in urine, our findings suggest that the kidney serves as a main site for its degradation.

Male Sprague Dawley rats (300 g) were infused by constant infusion into the jugular vein through 3-5 h with human leukocyte-derived interferon (HuIFN-alpha 10(6) units/h, 0.9 ml/h). Blood samples were collected every 20 min through the carotid. A steady-state level of interferon in serum (10(4) U/ml) was reached after 50 min of infusion, indicating fast removal of most of the infused material. Examination of interferon distribution in various tissues at the termination of the infusion revealed that over 85% of active interferon was found in the kidneys. No interferon was found in urine. Ligation of both kidneys resulted (after 2.5 h of infusion) in a ten-fold increase in interferon in serum and a minor increase in other tissues. No interferon was found in the kidneys. Simultaneous infusion of HuIFN-alpha, pepstatin and leupeptin (the well-known inhibitors of lysosomal proteinases) for 2.5 h had no effect on interferon concentration in serum and various tissues except in kidneys where a 5-fold increase was found. Mitochondrial-lysosomal fractions contained 55% of the total kidney interferon. Partial inhibition of proteolytic and HuIFN-alpha degrading activities at that fraction was also observed. These data, together with our previous findings of accumulation of injected interferon in the mitochondrial-lysosomal fraction of rats and monkeys' kidney cells, provide a further evidence for the main role of the kidney in HuIFN-alpha degradation.

In order to follow the catabolic fate of interferon in the body, [125I]-labeled human alpha interferon was infused into rats. Interferon activity and TCA-precipitable radioactivity in the blood reached a steady state after 60 min of infusion, while TCA-soluble radioactivity continued to increase during the entire infusion period. A massive accumulation of both active interferon and degradation products of interferon were found in the kidney at the end of the infusion. Most of the interferon activity was found in the mitochondrial-lysosomal fraction, while most of the interferon degradation products were found in the supernatant fraction. Ligation of the kidney's blood vessels resulted in a large increase of interferon activity in the blood. Only a small increase was found in other organs, with no increase at all in radioactive degradation products. Our results suggest the kidney to be the main site of interferon catabolism. Low molecular weight degradation products are excreted from the kidney into the blood and are at least partially secreted in the urine and also taken up by other organs.