Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine.

Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine Xiangyang Chi, Jianmin Li, Weicen Liu, Xiaolin Wang, Kexin Yin, Ju Liu, Xiaodong Zai, Liangliang Li, Xiaohong Song, Jun Zhang, Xiaopeng Zhang, Ying Yin, Ling Fu, Junjie Xu, Changming Yu, Wei Chen Laboratory of Vaccine and Antibody Engineering, Beijing Institute of Biotechnology, Beijing, China The anthrax protective antigen (PA) is the central component of the three-part anthrax toxin, and it is the primary immunogenic component in the approved AVA anthrax vaccine and the “next-generation” recombinant PA (rPA) anthrax vaccines. Animal models have indicated that PA-specific antibodies (AB) are sufficient to protect against infection with Bacillus anthracis. In this study, we investigated the PA domain specificity, affinity, mechanisms of neutralization, and synergistic effects of PA-specific antibodies from a single donor following vaccination with the rPA vaccine. Antibody-secreting cells were isolated 7 days after the donor received a boost vaccination, and 34 fully human monoclonal antibodies (hMAb) were identified. Clones 8H6, 4A3, and 22F1 were able to neutralize lethal toxin (LeTx) both in vitro and in vivo. Clone 8H6 neutralized LeTx by preventing furin cleavage of PA in a dose-dependent manner. Clone 4A3 enhanced degradation of nicked PA, thereby interfering with PA oligomerization. The mechanism of 22F1 is still unclear. A fourth clone, 2A6, that was protective only in vitro was found to be neutralizing in vivo in combination with a toxin-enhancing antibody, 8A7, which binds to domain 3 of PA and PA oligomers. These results provide novel insights into the antibody response elicited by the rPA vaccine and may be useful for PA-based vaccine and immunotherapeutic cocktail design. B acillus anthracis, the causative agent of anthrax, has long been considered a serious public health threat, particularly after the bioterrorism attacks that occurred after 11 September 2001. B. anthracis secretes a tripartite toxin that consists of protective antigen (PA; 83 kDa), lethal factor (LF; 85 kDa), and edema factor (EF; 89 kDa). LF is a Zn2⫹-dependent metalloprotease (1) that combines with PA to form lethal toxin (LeTx), which causes death when injected into animals intravenously. EF is a Ca2⫹-dependent adenylyl cyclase enzyme (2) that combines with PA to form edema toxin (EdTx), which causes edema at the site of inoculation. PA by itself is not known to have pathogenic effects but is considered the central component of the anthrax toxin. It primarily functions as a vehicle mediating the cellular uptake of EF and LF. PA binds to cell surface receptors (CMG-2/TEM-8) on target host cells (3, 4). Following binding, PA is cleaved by furin-like protease produced by the target cells to PA20, a 20-kDa peptide that is released into the surroundings. The remaining fragment, PA63 (also known as nicked/activated PA63), remains bound to the receptor and forms a heptamer or octamer that can translocate as many as three molecules of EF or LF from the cell surface into the cytosol via endocytosis, leading to the biological effects of EF and LF (5). Vaccination against PA is sufficient to elicit immune protection. PA is the primary immunogenic component in the anthrax vaccine adsorbed (AVA) formulation that is currently licensed in the United States for human use. Although effective, AVA requires a much longer time (6 months) to produce protective immunity than that required for any effective postexposure anthrax prophylaxis, and the exact antigenic composition of the vaccine remains unknown and varies between batches (6, 7). The only immunogenic component in the “next-generation” anthrax vaccine is recombinant PA (rPA). The rPA vaccine can potentially elicit a faster protective immune response to be effective in postexposure cases and be produced in more homogenous standard formula- May 2015 Volume 22 Number 5 tions (7). Clinical trials have demonstrated the safety and immunogenicity of the rPA vaccine (8, 9). Vaccination with rPA is known to elicit a polyclonal antibody (Ab) response. Most of the studies that have been conducted characterize the antibody responses induced by the AVA vaccine in humans (10). To date, there have not been very many studies to characterize the human antibody responses to the rPA vaccine. The conventional treatment following potential exposure to aerosolized B. anthracis spores was antibiotics combined with PAbased anthrax vaccine. While antibiotics are effective in killing bacteria, they are unable to clear toxin components from the bloodstream (11). Passive immunization with monoclonal antibodies (MAbs) against toxin components has been shown to be highly protective in postexposure cases, particularly when combined with antibiotics (12, 13). Characterizing the protective rPA vaccine-induced antibody response in humans may identify naturally occurring neutralizing antibodies that could be used therapeutically. Received 10 December 2014 Returned for modification 8 January 2015 Accepted 3 March 2015 Accepted manuscript posted online 18 March 2015 Citation Chi X, Li J, Liu W, Wang X, Yin K, Liu J, Zai X, Li L, Song X, Zhang J, Zhang X, Yin Y, Fu L, Xu J, Yu C, Chen W. 2015. Generation and characterization of human monoclonal antibodies targeting anthrax protective antigen following vaccination with a recombinant protective antigen vaccine. Clin Vaccine Immunol 22:553–560. doi:10.1128/CVI.00792-14. Editor: D. L. Burns Address correspondence to Changming Yu, , or Wei Chen, . Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/CVI.00792-14 Clinical and Vaccine Immunology cvi.asm.org 553 Chi et al. Most of the monoclonal antibodies neutralizing PA-mediated toxicity have been produced in murine sources (14, 15), although some protective monoclonal antibodies against PA have been developed in chimpanzees, transgenic mice, and humans (16–18). The first Food and Drug Administration-approved monoclonal antibody against PA is fully human (19). Identification and characterization of novel protective anti-PA antibodies could contribute to the development of additional therapeutics for treating anthrax in patients. In this study, we assessed the neutralizing potential of a large panel of monoclonal antibodies generated from an individual donor immunized with the rPA vaccine. We further characterized the domain specificities, affinities, mechanisms of neutralization, and potential synergistic effects of the protective human monoclonal antibodies (hMAbs). Overall, our results provide novel insights into the donor’s antibody responses to the rPA vaccine and information about the design of PA-based vaccines and immunotherapeutic cocktails of hMAbs. MATERIALS AND METHODS Recombinant anthrax toxins. Recombinant protective antigen (rPA) and lethal factor (rLF) were expresse (...truncated)