Characterization of the adaptive immune response of donors receiving live anthrax vaccine

Live anthrax vaccine containing spores from attenuated strains STI-1 of Bacillus anthracis is used in Russia and former CIS (Commonwealth of Independent States) to prevent anthrax. In this paper we studied the duration of circulation of antibodies specific to spore antigens, the protective antigen (PA), the lethal factor (LF) and their domains (D) in donors’ blood at different times after their immunization with live anthrax vaccine. The relationship between the toxin neutralization activity level and the level of antibodies to PA, LF and their domains was tested. The effect of age, gender and number of vaccinations on the level of adaptive post-vaccination immune response has been studied. It was shown that antibodies against PA-D1 circulate in the blood of donors for 1 year or more after immunization with live anthrax vaccine. Antibodies against all domains of LF and PA-D4 were detected in 11 months after vaccination. Antibodies against the spores were detected in 8 months after vaccination. A moderate positive correlation was found between the titers of antibodies to PA, LF, or their domains, and the TNA of the samples of blood serum from the donors.

Using a Syrian (Golden) Hamster Biological Model for the Evaluation of Recombinant Anthrax Vaccines

In this paper, we demonstrate that a Syrian hamster biological model can be applied to the study of recombinant anthrax vaccines. We show that double vaccination with recombinant proteins, such as protective antigen (PA) and fusion protein LF1PA4, consisting of lethal factor I domain (LF) and PA domain IV, leads to the production of high titers of specific antibodies and to protection from infection with the toxicogenic encapsulated attenuated strain B. anthracis 71/12. In terms of antibody production and protection, Syrian hamsters were much more comparable to guinea pigs than mice. We believe that Syrian hamsters are still underestimated as a biological model for anthrax research, and, in some cases, they can be used as a replacement or at least as a complement to the traditionally used mouse model.

Solution Structures of Bacillus anthracis Protective Antigen Proteins Using Small Angle Neutron Scattering and Protective Antigen 63 Ion Channel Formation Kinetics

We are studying the structures of bacterial toxins that form ion channels and enable macromolecule transport across membranes. For example, the crystal structure of the Staphylococcus aureus α-hemolysin (α-HL) channel in its functional state was confirmed using neutron reflectometry (NR) with the protein reconstituted in membranes tethered to a solid support. This method, which provides sub-nanometer structural information, could also test putative structures of the Bacillus anthracis protective antigen 63 (PA63) channel, locate where B. anthracis lethal factor and edema factor toxins (LF and EF, respectively) bind to it, and determine how certain small molecules can inhibit the interaction of LF and EF with the channel. We report here the solution structures of channel-forming PA63 and its precursor PA83 (which does not form channels) obtained with small angle neutron scattering. At near neutral pH, PA83 is a monomer and PA63 a heptamer. The latter is compared to two cryo-electron microscopy structures. We also show that although the α-HL and PA63 channels have similar structural features, unlike α-HL, PA63 channel formation in lipid bilayer membranes ceases within minutes of protein addition, which currently precludes the use of NR for elucidating the interactions between PA63, LF, EF, and potential therapeutic agents.

Multiplexed, High-Sensitivity Measurements of Antibody Affinity Using Interferometric Reflectance Imaging Sensor

Anthrax lethal factor (LF) is one of the enzymatic components of the anthrax toxin responsible for the pathogenic responses of the anthrax disease. The ability to screen multiplexed ligands against LF and subsequently estimate the effective kinetic rates (kon and koff) and complementary binding behavior provides critical information useful in diagnostic and therapeutic development for anthrax. Tools such as biolayer interferometry (BLI) and surface plasmon resonance imaging (SPRi) have been developed for this purpose; however, these tools suffer from limitations such as signal jumps when the solution in the chamber is switched or low sensitivity. Here, we present multiplexed antibody affinity measurements obtained by the interferometric reflectance imaging sensor (IRIS), a highly sensitive, label-free optical biosensor, whose stability, simplicity, and imaging modality overcomes many of the limitations of other multiplexed methods. We compare the multiplexed binding results obtained with the IRIS system using two ligands targeting the anthrax lethal factor (LF) against previously published results obtained with more traditional surface plasmon resonance (SPR), which showed consistent results, as well as kinetic information previously unattainable with SPR. Additional exemplary data demonstrating multiplexed binding and the corresponding complementary binding to sequentially injected ligands provides an additional layer of information immediately useful to the researcher.

Epitopedia: identifying molecular mimicry of known immune epitopes

MotivationUpon infection, pathogen epitopes stimulate the host’s immune system to produce antibodies targeting the pathogen. Molecular mimicry (structural similarity) between an infecting pathogen and host proteins or pathogenic proteins the host has previously encountered can impact the immune response of the host. The ability to identify potential molecular mimicry for a pathogen can illuminate immune effects with importance to pathogen treatment and vaccine design.SummaryEpitopedia allows for identification of regions with three-dimensional molecular mimicry between a protein in a pathogen with known epitopes in the host.ResultsSARS-CoV-2 Spike returns molecular mimicry with 14 different epitopes including integrin beta-1 from Homo sapiens, lethal factor precursor from Bacillus anthracis, and pollen allergen Phl p 2 from Timothy grass.AvailabilityEpitopedia is primarily written in Python and relies on established software and databases. Epitopedia is available at https://github.com/cbalbin-FIU/Epitopedia under the opensource MIT license and is also packaged as a docker container at https://hub.docker.com/r/cbalbin/[email protected], [email protected]

Anthrax toxin translocation complex reveals insight into the lethal factor unfolding and refolding mechanism

Translocation is essential to the anthrax toxin mechanism. Protective antigen (PA), the binding component of this AB toxin, forms an oligomeric pore that translocates lethal factor (LF) or edema factor, the active components of the toxin, into the cell. Structural details of the translocation process have remained elusive despite their biological importance. To overcome the technical challenges of studying translocation intermediates, we developed a method to immobilize, transition, and stabilize anthrax toxin to mimic important physiological steps in the intoxication process. Here, we report a cryoEM snapshot of PApore translocating the N-terminal domain of LF (LFN). The resulting 3.3 Å structure of the complex shows density of partially unfolded LFN near the canonical PApore binding site. Interestingly, we also observe density consistent with an α helix emerging from the 100 Å β barrel channel suggesting LF secondary structural elements begin to refold in the pore channel. We conclude the anthrax toxin β barrel aids in efficient folding of its enzymatic payload prior to channel exit. Our hypothesized refolding mechanism has broader implications for pore length of other protein translocating toxins.

Probing Conformational Dynamics by Protein Contact Networks: Comparison with NMR Relaxation Studies and Molecular Dynamics Simulations

Protein contact networks (PCNs) have been used for the study of protein structure and function for the past decade. In PCNs, each amino acid is considered as a node while the contacts among amino acids are the links/edges. We examined the possible correlation between the closeness centrality measure of amino acids within PCNs and their mobility as known from NMR spin relaxation experiments and molecular dynamic (MD) simulations. The pivotal observation was that plasticity within a protein stretch correlated inversely to closeness centrality. Effects on protein conformational plasticity caused by the formation of disulfide bonds or protein–protein interactions were also identified by the PCN analysis measure closeness centrality and the hereby introduced percentage of closeness centrality perturbation (% CCP). All the comparisons between PCN measures, NMR data, and MDs were performed in a set of proteins of different biological functions and structures: the core protease domain of anthrax lethal factor, the N-terminal RING domain of E3 Ub ligase Arkadia, the reduced and oxidized forms of human thioredoxin 1, and the ubiquitin molecules (Ub) of the catalytic Ub–RING–E3–E2–Ub complex of E3 ligase Ark2.The graph theory analysis of PCNs could thus provide a general method for assessing the conformational dynamics of free proteins and putative plasticity changes between different protein forms (apo/complexed or reduced/oxidized).