Localization and characterization of a specific linear epitope of the Brucella DnaK protein

ABSTRACT Due to the potential use of ricin and other fast-acting toxins as agents of bioterrorism, there is an urgent need for the development of safe and effective antitoxin vaccines. A candidate ricin subunit vaccine (RiVax) consisting of a recombinant attenuated enzymatic A chain (RTA) has been shown to elicit protective antitoxin antibodies in mice and rabbits and is currently being tested in phase I human clinical trials. However, evaluation of the efficacy of this vaccine for humans is difficult for a number of reasons, including the fact that the key neutralizing B-cell epitopes on RTA have not been fully defined. Castelletti and colleagues (Clin. Exp. Immunol. 136:365-372, 2004) recently identified a linear epitope on RTA, spanning residues L161 to I175, as a primary target of serum antibodies derived from humans who had been treated with ricin immunotoxin. While affinity-purified polyclonal IgG antibodies against this region of RTA were capable of neutralizing ricin in vitro, their capacity to confer protection against ricin challenge in vivo was not determined. In this report, we describe the production and characterization of GD12, a murine monoclonal IgG1 antibody specifically directed against residues 163 to 174 (TLARSFIICIQM) of RTA. GD12 bound ricin holotoxin with high affinity (KD [dissociation constant], 2.9 × 10−9 M) and neutralized it with a 50% inhibitory concentration of ∼0.25 μg/ml, as determined by a Vero cell-based cytotoxicity assay. Passive administration of GD12 was sufficient to protect BALB/c mice against intraperitoneal and intragastric ricin challenges. These data are important in terms of vaccine development, since they firmly establish that preexisting serum antibodies directed against residues 161 to 175 on RTA are sufficient to confer both systemic and mucosal immunity to ricin. The potential of GD12 to serve as a therapeutic following ricin challenge was not explored in this study.

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Characterization of the dnaK Multigene Family in the Cyanobacterium Synechococcus sp. Strain PCC7942

Journal of Bacteriology ◽

10.1128/jb.183.4.1320-1328.2001 ◽

2001 ◽

Vol 183 (4) ◽

pp. 1320-1328 ◽

Cited By ~ 31

Author(s):

Kaori Nimura ◽

Hideo Takahashi ◽

Hirofumi Yoshikawa

Keyword(s):

Heat Shock ◽

Expression System ◽

Normal Growth ◽

Permissive Temperature ◽

E Coli ◽

Growth Deficiency ◽

Varied Response ◽

Synechococcus Sp ◽

Dnak Protein

ABSTRACT The cyanobacterium Synechococcus sp. strain PCC7942 has three dnaK homologues (dnaK1, dnaK2, anddnaK3), and a gene disruption experiment was carried out for each dnaK gene by inserting an antibiotic resistance marker. Our findings revealed that DnaK1 was not essential for normal growth, whereas DnaK2 and DnaK3 were essential. We also examined the effect of heat shock on the levels of these three DnaK and GroEL proteins and found a varied response to heat shock, with levels depending on each protein. The DnaK2 and GroEL proteins exhibited a typical heat shock response, that is, their synthesis increased upon temperature upshift. In contrast, the synthesis of DnaK1 and DnaK3 did not respond to heat shock; in fact, the level of DnaK1 protein decreased. We also analyzed the effect of overproduction of each DnaK protein in Escherichia coli cells using an inducible expression system. Overproduction of DnaK1 or DnaK2 resulted in defects in cell septation and formation of cell filaments. On the other hand, overproduction of DnaK3 did not result in filamentous cells; rather a swollen and twisted cell morphology was observed. When expressed in anE. coli dnaK756 mutant, dnaK2 could suppress the growth deficiency at the nonpermissive temperature, whilednaK1 and dnaK3 could not suppress this phenotype. On the contrary, overproduction of DnaK1 or DnaK3 resulted in growth inhibition at the permissive temperature. These results suggest that different types of Hsp70 in the same cellular compartment have specific functions in the cell.

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