Nehal Mohamed

A High-Affinity Monoclonal Antibody to Anthrax Protective Antigen Passively Protects Rabbits before and after Aerosolized Bacillus anthracis Spore Challenge

ABSTRACT We have developed a therapeutic for the treatment of anthrax using an affinity-enhanced monoclonal antibody (ETI-204) to protective antigen (PA), which is the central cell-binding component of the anthrax exotoxins. ETI-204 administered preexposure by a single intravenous injection of a dose of between 2.5 and 10 mg per animal significantly protected rabbits from a lethal aerosolized anthrax spore challenge (∼60 to 450 times the 50% lethal dose of Bacillus anthracis Ames). Against a similar challenge, ETI-204 administered intramuscularly at a 20-mg dose per animal completely protected rabbits from death (100% survival). In the postexposure setting, intravenous administration of ETI-204 provided protection 24 h (8 of 10) and 36 h (5 of 10) after spore challenge. Administration at 48 h postchallenge, when 3 of 10 animals had already succumbed to anthrax infection, resulted in the survival of 3 of 7 animals (43%) for the duration of the study (28 days). Importantly, surviving ETI-204-treated animals were free of bacteremia by day 10 and remained so until the end of the studies. Only 11 of 51 ETI-204-treated rabbits had positive lung cultures at the end of the studies. Also, rabbits that were protected from inhalational anthrax by administration of ETI-204 developed significant titers of PA-specific antibodies. Presently, the sole therapeutic regimen available to treat infection by inhalation of B. anthracis spores is a 60-day course of antibiotics that is effective only if administered prior to or shortly after exposure. Based upon results reported here, ETI-204 is an effective therapy for prevention and treatment of inhalational anthrax.

Enhancement of Anthrax Lethal Toxin Cytotoxicity: a Subset of Monoclonal Antibodies against Protective Antigen Increases Lethal Toxin-Mediated Killing of Murine Macrophages

ABSTRACT We investigated the ability of using monoclonal antibodies (MAbs) against anthrax protective antigen (PA), an anthrax exotoxin component, to modulate exotoxin cytotoxic activity on target macrophage cell lines. Anthrax PA plays a critical role in the pathogenesis of Bacillus anthracis infection. PA is the cell-binding component of the two anthrax exotoxins: lethal toxin (LeTx) and edema toxin. Several MAbs that bind the PA component of LeTx are known to neutralize LeTx-mediated killing of target macrophages. Here we describe for the first time an overlooked population of anti-PA MAbs that, in contrast, function to increase the potency of LeTx against murine macrophage cell lines. The results support a possible mechanism of enhancement: binding of MAb to PA on the macrophage cell surface stabilizes the PA by interaction of MAb with macrophage Fcγ receptors. This results in an increase in the amount of PA bound to the cell surface, which in turn leads to an enhancement in cell killing, most likely due to increased internalization of LF. Blocking of PA-receptor binding eliminates enhancement by MAb, demonstrating the importance of this step for the observed enhancement. The additional significance of these results is that, at least in mice, immunization with PA appears to elicit a poly-clonal response that has a significant prevalence of MAbs that enhance LeTx-mediated killing in macrophages.

Novel experimental study of receptor-mediated bacterial adhesion under the influence of fluid shear.

Dynamic adhesion of cells to surfaces is a vital step in a variety of biochemical and physiological phenomena. Bacterial adhesion is responsible not only for problems associated with biofouling and biofilm formation in the biochemical industry but also in the initiation of certain infectious diseases. In this study, we report the effect of critical parameters, such as receptor and ligand densities and shear rate, on receptor-mediated dynamic bacterial adhesion. Adhesion of a pathogenic strain of Staphylococcus aureus to immobilized collagen was studied. The receptor density on the cell surface was varied by harvesting cells at different growth times and was quantified using flow cytometry. Dynamic adhesion experiments were conducted over a range of physiologically relevant shear rates (50 to 1500 s(-1)) using a parallel-plate flow chamber. Video microscopy coupled with digital image processing was employed to quantify adhesion. A semiquantitative comparison between experimental results and theoretical data obtained using a previously proposed mathematical model was also performed. The results suggest that dynamic adhesion is dependent on receptor density and shear rate, but independent of ligand density. This report demonstrates the feasibility of using bacteria to study fundamental aspects of receptor-mediated dynamic adhesion.

Shear Stress Affects the Kinetics of Staphylococcus aureus Adhesion to Collagen

Staphylococcus aureus is a major human pathogen that has been shown to bind collagen under static conditions. However, many staphylococcal infections are hematogenously acquired and adhesion events may be influenced by shear stress. In this study, we used a dynamic experimental system consisting of a parallel‐plate perfusion chamber and phase‐contrast video microscope to study the effects of shear stress on the adhesion kinetics of intact S. aureus to collagen surfaces in vitro. The adhesion of S. aureus Phillips to collagen types I, II, and IV was investigated over a physiologically relevant range of wall shear stresses at 37 °C. S. aureus PH100, a collagen adhesin‐deficient mutant strain, was used as a control strain for the experiments. We found that S. aureus Phillips could adhere to collagens I, II, and IV at wall shear stresses less than 15 dyn/cm2 and that the kinetics of the adhesion process were wall shear stress‐dependent. Similar studies with PH100 demonstrated that these cells are unable to adhere firmly to collagen surfaces. Transient interactions between PH100 and the collagen surfaces were observed at low levels of shear stress suggesting that S. aureus may also interact with collagen by an alternative mechanism that does not lead to firm adhesion.