Tamar Bino

Generation of Yersinia pestis Attenuated Strains by Signature-Tagged Mutagenesis in Search of Novel Vaccine Candidates

ABSTRACT In a search for novel attenuated vaccine candidates for use against Yersinia 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 virulent Y. pestis Kimberley53 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 10−5 to 10−7 to 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, and greA) or for hypothetical polypeptides, as well as for the virulence regulator gene lcrF. One of the avirulent mutant strains (LD50, >107 CFU) was found to be disrupted in the pcm locus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcm mutant 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.

Electrostatic Attraction by Surface Charge does not Contribute to the Catalytic Efficiency of Acetylcholinesterase

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.

Effective Protective Immunity to Yersinia pestis Infection Conferred by DNA Vaccine Coding for Derivatives of the F1 Capsular Antigen

ABSTRACT Three plasmids expressing derivatives of the Yersinia pestis capsular F1 antigen were evaluated for their potential as DNA vaccines. These included plasmids expressing the full-length F1, F1 devoid of its putative signal peptide (deF1), and F1 fused to the signal-bearing E3 polypeptide of Semliki Forest virus (E3/F1). Expression of these derivatives in transfected HEK293 cells revealed that deF1 is expressed in the cytosol, E3/F1 is targeted to the secretory cisternae, and the nonmodified F1 is rapidly eliminated from the cell. Intramuscular vaccination of mice with these plasmids revealed that the vector expressing deF1 was the most effective in eliciting anti-F1 antibodies. This response was not limited to specific mouse strains or to the mode of DNA administration, though gene gun-mediated vaccination was by far more effective than intramuscular needle injection. Vaccination of mice with deF1 DNA conferred protection against subcutaneous infection with the virulent Y. pestis Kimberley53 strain, even at challenge amounts as high as 4,000 50% lethal doses. Antibodies appear to play a major role in mediating this protection, as demonstrated by passive transfer of anti-deF1 DNA antiserum. Taken together, these observations indicate that a tailored genetic vaccine based on a bacterial protein can be used to confer protection against plague in mice without resorting to regimens involving the use of purified proteins.

Structural Modifications of the Ω Loop in Human Acetylcholinesterase

Conformational mobility of the surface Ω loop (Cys‐69‐Cys‐96) in human acetylcholinesterase (HuAChE) was recently implicated in substrate accessibility to the active center and in the mechanism of allosteric modulation of enzymatic activity. We therefore generated and kinetically evaluated the following modifications or replacements in HuAChE: (a) residues at the loop ends, (b) residues involved in putative hydrogen‐bond interactions within the loop and between the loop and the protein core, (c) ChEs conserved proline residues within the loop and (d) a deletion of a conserved segment of 5 residues. All the residue replacements, including those of the prolines, had either limited or no effect on enzyme reactivity. These results suggest that unlike the case of lipase, the Ω loop in the HuAChE is not involved in large lid‐like displacements. In cases where modifications of the loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp‐86 supporting the proposed coupling between the structure of the Ω loop and the positioning of the Trp‐86 indole moiety, in catalytic activity and in allosterism.

Vaccination with plasmid DNA expressing the Yersinia pestis capsular protein F1 protects mice against plague.

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.

Mechanism of target cell recognition by CD3− LGL: I. Development of a monoclonal antibody to a K562-associated target cell antigen

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.

Alphavirus Hybrid Virion Vaccines

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.

What can be Learned from the use of HuAChE Mutants for Evaluation of Potential Alzheimer’s Drugs

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.

Compatibility of Structures Inferred from Mutagenesis and from X-Ray Crystallography for Various AChE Complexes

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.

Molecular Aspects of Catalysis and of Allosteric Regulation of Aceytlcholinesterases

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).