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Infect. Immun. doi:10.1128/IAI.01722-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Enhancement of antibody responses to protective antigen (PA(IV)) of Bacillus anthracis using calreticulin as a chimeric molecular adjuvant

Laboratory Of Infection and Immunology, Graduate School of Medicine, Korea University, Seoul, South Korea, and Departments of Pathology, Obstetrics and Gynecology, Oncology, Molecular Microbiology and Immunology and Otolaryngology/Head and Neck Surgery, The Johns Hopkins Medical Institutions, Baltimore, Maryland, USA

^* To whom correspondence should be addressed. Email: wutc{at}jhmi.edu. twkim0421{at}korea.com.

	Abstract

The generation of protective humoral immune responses against the receptor binding domain of protective antigen (PA(dIV)) of Bacillus anthracis represents a plausible approach against anthrax toxin. In the current study, we have developed a naked DNA vaccine encoding calreticulin (CRT) linked to the receptor-binding domain (domain IV) of the protective antigen (PA) of Bacillus anthracis (CRT/PA(dIV)). We transfected a human embryonic kidney cell line (HEK293) with CRT/PA(dIV) DNA and performed Western blot and confocal microscopy analysis. We found that linkage of CRT to PA(dIV) targets PA(dIV) to the ER, resulting in secretion of the chimeric CRT/PA(dIV) protein. We then evaluated the ability of CRT/PA(dIV) DNA to generate PA(dIV)-specific antibody responses and protective immunity against lethal anthrax toxin (PA plus LF) challenge. We found that mice immunized with CRT/PA(dIV) DNA were capable of rapidly inducing significantly higher PA(dIV)-specific antibody responses compared to mice immunized with PA(dIV) DNA alone. Furthermore, we observed that this enhanced antibody response generated by CRT/PA(dIV) DNA was CD4-dependant since CD4 knockout mice demonstrated significant reduction in antibody responses. In addition, analysis of the titers and avidity maturation of the induced PA-specific antibodies revealed that vaccination with CRT/PA(dIV) DNA vaccine accelerated the avidity maturation of antibodies to PA(dIV) compared to vaccination with PA(dIV) DNA. Importantly, the enhanced antibody responses correlated to protective immunity against lethal anthrax toxin challenge. Thus, DNA vaccines encoding CRT linked to PA(dIV) may dramatically enhance PA-specific protective antibody responses. Our results have significant clinical applications for biodefense against anthrax toxin.