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

Abstract

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.

Quantitative Determination of Lethal Toxin Proteins in Culture Supernatant of Human Live Anthrax Vaccine Bacillus anthracis A16R

“…The rPA and rLF proteins at fixed amounts (50 ng) and LT heptamers complexes were also detected in the co-IP assay as a control. The LT heptamers complexes were prepared as described previously [ 21 ]. PA83 was treated with trypsin at a final trypsin/PA weight ratio of 1:1000 for 30 min at room temperature.…”

“…Various studies carried out with anthrax vaccines in different animal models indicate the relevance of PA as a key component of the vaccine [ 16 , 18 ]. Antibodies generated against PA, especially those that have anthrax toxin neutralization activity, have been established as being critical for immunity to anthrax [ 19 , 20 , 21 ].…”

Potent neutralizing monoclonal antibodies against Ebola virus isolated from vaccinated donors

“…After cell sorting, RT-PCR was immediately carried out using the SuperScript™ III First-Strand Synthesis System (18080051; Invitrogen, Thermo Fisher Scientific), and followed by a nested PCR using TransStart Taq DNA polymerase (AP141; TransGen) to amplify cDNAs encoding VH and VL of antibodies from single cells. 47,48 VH or VL genes were then constructed into linear cassettes containing a CMV promoter, Ig leader fragments, CH or CL of IgG1, and poly-A tail to obtain full-length Ig heavy and light chains as previously described. 31 Quick expression of antibodies using linear cassettes Human embryonic kidney HEK293 T cells (ATCC® CRL-11268; ATCC) were seeded into 96-well culture plates at 2 × 10 4 cells/well 24 h before transfection and cultured in 200 μL Dulbecco's Modified Eagle Medium (DMEM) (C11995500; Gibco) supplemented with 10% FBS, 100 mg/ mL streptomycin, and 100 I.U./mL penicillin at 37°C and 5% CO 2 .…”

“…After blocking, biotinylated antibodies at a final EC 50 calculated as above were mixed with 5 μg/mL of competitors (about 100-fold molar excess), and the mixture was added to coated plates, followed by incubation for 1 h at 37°C. An antibody specific to anthrax protective antigen, 8A7, 48 was used as an irrelevant competitor. Biotinylated antibodies bound to GPΔMuc were detected using HRP-conjugated streptavidin (SNN1004; Thermo Fisher Scientific) at 450/630 nm.…”

Design and Testing of Novel Anthrax Vaccines Utilizing a Tobacco Mosaic Virus Expression System

“…This fact is supported by a number of other studies that report mAbs that bind domain III but on their own are incapable of neutralizing anthrax LT. Antibody 2-A7, that was found to bind amino acids G532 to Q543, did not protect mice against LT challenge 80 . Fisher 344 Rats challenged with LT and administered domain III binding antibody 8A7 at a 1:1 antibody to PA molar ratio also all died 87 . However, when 9 μg of 8A7 was administered in combination with 9 μg of 2A6…”