Potency and stability of a trivalent, catalytically inactive vaccine against botulinum neurotoxin serotypes C, E and F (triCEF).

Botulism is an acute neuroparalytic affliction of the motor and autonomic neurons caused by the toxins produced from Clostridium botulinum and related bacterial strains. The botulinum neurotoxins, or BoNTs, consist of a phylogenetically diverse group of highly potent protein toxins. Current medical interventions for confirmed cases of botulism are limited to immediate administration of antitoxins and respiratory support. There is currently no licensed vaccine against botulism in the United States. The most widely distributed botulism vaccine was a pentavalent BoNT toxoid (PBT) against serotypes A-E administered until 2011 under an investigational new drug license. A binary vaccine composed of the recombinant, non-toxic, receptor binding domains (RBD) of serotypes/A1 and/B1 has completed a phase II clinical trial, but has yet to attain full licensure. We have previously published data demonstrating catalytically inactive, full length botulinum neurotoxin holoproteins (ciBoNT HPs) against serotypes/A1,/B1,/C1,/E1 and/F1 provide equivalent or superior potency against parental and dissimilar subtype toxins as compared the RBD vaccines. Here we describe the consistent potencies of the three independent lots each of ciBoNT/C1,/E1, and/F1 HPs against substantial monovalent challenges of the parental toxins. We also present data that a trivalent formulation of ciBoNT/C1,/E1 and/F1 (triCEF) maintains potency against both monovalent and polyvalent toxin challenges when stored as an adjuvanted vaccine at 4-8 °C for up to 2 years.

Recombinant Botulinum Neurotoxin Hc Subunit (BoNT Hc) and Catalytically Inactive Clostridium botulinum Holoproteins (ciBoNT HPs) as Vaccine Candidates for the Prevention of Botulism.

There are few available medical countermeasures against botulism and the discontinuation of the pentavalent botulinum toxoid vaccine by the Centers for Disease Control and Prevention in 2011 has resulted in the need for a safe and effective prophylactic alternative. Advances in genetic engineering have resulted in subsequent vaccine efforts being primarily focused on the production of highly purified recombinant protein antigens representing one or more domains of the botulinum neurotoxin. Recombinant subunit vaccines based on the carboxy one-third of the toxin (Hc) developed in our lab against serotypes A-F have been shown to be safe and effective. However, in response to the identification of an ever increasing number of BoNT subtypes with significant amino acid heterogeneity, we have developed catalytically inactive BoNT holoproteins (ciBoNT HPs) in an attempt to elicit greater protective immunity to address these toxin variants. Here we report the production of ciBoNT/B1 HP, ciBoNT/C1 HP, ciBoNT/E1 HP and ciBoNT/F1 HP and compare the immunological and protective abilities of ciBoNT HPs and BoNT/A Hc, BoNT/B Hc, BoNT/C Hc, BoNT/E Hc and BoNT/F Hc vaccines when challenged with homologous and heterologous toxins. Our results suggest the ciBoNT HP vaccines exhibit superior potency after single vaccinations but multiple vaccinations with BoNT/Hc antigens resulted in increased survival rates at the toxin challenge levels used.

A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model.

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant 'targeting agent' that binds a toxin at two unique sites and a 'clearing Ab' that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab V(H) (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit.

Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins.

Recombinant botulinum Hc (rBoNT Hc) vaccines for serotypes C1 and D were produced in the yeast Pichia pastoris and used to determine protection against four distinct BoNT C and D toxin subtypes. Mice were vaccinated with rBoNT/C1 Hc, rBoNT/D Hc, or with a combination of both vaccines and challenged with BoNT C1, D, C/D, or D/C toxin. Mice receiving monovalent vaccinations were partially or completely protected against homologous toxin and not protected against heterologous toxin. Bivalent vaccine candidates completely survived challenges from all toxins except D/C toxin. These results indicate the recombinant C1 and D Hc vaccines are not only effective in a monovalent formula but offer complete protection against both parental and C/D mosaic toxin and partial protection against D/C mosaic toxin when delivered as a bivalent vaccine.

Characterization of an Escherichia coli mutant MutY with a cysteine to alanine mutation at the iron-sulfur cluster domain.

Escherichia coli MutY is an adenine and a weak guanine DNA glycosylase involved in reducing mutagenic effects of 7,8-dihydro-8-oxoguanine (8-oxoG). The [4Fe-4S] cluster of MutY is ligated by four conserved cysteine residues and has been shown to be important in substrate recognition. Here, we show that the C199A mutant MutY is very insoluble and can be denatured and renatured to regain activity only if iron and sulfur are present in the renaturation steps. The solubility of C199A-MutY can be improved substantially as a fusion protein containing streptococcal protein G (GB1 domain) at its N-terminus. Here, we describe the first biochemical characterization of the purified GB1-C199A-MutY protein which contains a [3Fe-4S] cluster. The apparent dissociation constant (K(d)) values of GB1-C199A-MutY with both A/G and A/8-oxoG mismatches are slightly higher than that of the wild-type protein. The DNA glycosylase activity of GB1-C199A-MutY is comparable to that of the wild-type enzyme. Interestingly, the major difference between the C199A-MutY and wild-type proteins is their trapping activities (formation of Schiff base intermediates). The GB1-C199A-MutY mutant has a weaker trapping activity than the wild-type enzyme. Importantly, highly expressed GB1-C199A-MutY and untagged C199A-MutY can complement mutY mutants; however, GB1-C199A-MutY and untagged C199A-MutY cannot complement mutY mutants in vivo when both proteins are poorly expressed. Therefore, an intact [4Fe-4S] cluster domain is critical for MutY stability and activity.

The Assessment of Goal Commitment: A Measurement Model Meta-Analysis.

Goals are central to current treatments of work motivation, and goal commitment is a critical construct in understanding the relationship between goals and performance. Inconsistency in the measurement of goal commitment hindered early research in this area but the nine-item, self-report scale developed by Hollenbeck, Williams, and Klein (1989b), and derivatives of that scale, have become the most commonly used measures of goal commitment. Despite this convergence, a few authors, based on small sample studies, have raised questions about the dimensionality of this measure. To address the conflicting recommendations in the literature regarding what items to use in assessing goal commitment, the current study combines the results of 17 independent samples and 2918 subjects to provide a more conclusive assessment by combining meta-analytic and multisample confirmatory factor analytic techniques. This effort reflects the first combined use of these techniques to test a measurement model and allowed for the creation of a database substantially larger than that of previously factor analyzed samples containing these scale items. By mitigating sampling error, the results clarified a number of debated issues that have arisen out of previous small sample factor analyses and revealed a five-item scale that is unidimensional and equivalent across measurement timing, goal origin, and task complexity. It is recommended that this five-item scale be used in future research assessing goal commitment. Copyright 2001 Academic Press.