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Enhancement of Antibody Responses to Bacillus anthracis Protective Antigen Domain IV by Use of Calreticulin as a Chimeric Molecular Adjuvant

Yong Sung Park,^1, Jin Hyup Lee,^2, Chien-Fu Hung,² T.-C. Wu,^2,3,4,5,6* and Tae Woo Kim^1*

Laboratory of Infection and Immunology, Graduate School of Medicine, Korea University, Seoul, South Korea,¹ Departments of Pathology,² Obstetrics and Gynecology,³ Oncology,⁴ Molecular Microbiology and Immunology,⁵ Otolaryngology/Head and Neck Surgery, The Johns Hopkins Medical Institutions, Baltimore, Maryland⁶

Received 21 December 2007/ Returned for modification 4 February 2008/ Accepted 7 February 2008

The generation of protective humoral immune responses against the receptor-binding domain (domain IV) 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 PA(dIV) of Bacillus anthracis [CRT/PA(dIV)]. We transfected a human embryonic kidney cell line (HEK 293) with CRT/PA(dIV) DNA and performed Western blotting and confocal microscopy analysis. We found that linkage of CRT to PA(dIV) targets PA(dIV) to the endoplasmic reticulum, 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 lethal factor) challenge. We found that mice immunized with CRT/PA(dIV) DNA were capable of rapidly inducing significantly higher PA(dIV)-specific antibody responses than mice immunized with PA(dIV) DNA alone. Furthermore, we observed that this enhanced antibody response generated by CRT/PA(dIV) DNA was CD4 dependent, since CD4 knockout mice demonstrated a 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.

Published ahead of print on 19 February 2008.

Editor: S. R. Blanke

Yong Sung Park and Jin Hyup Lee contributed equally to this work.