Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.

Ricin is a potent toxin associated with bioterrorism for which no vaccine or specific countermeasures are currently available. A stable, non-toxic and immunogenic recombinant ricin A-chain vaccine (RTA 1-33/44-198) has been developed by protein engineering. We identified optimal formulation conditions for this vaccine under which it remained stable and potent in storage for up to 18 months, and resisted multiple rounds of freeze-thawing without stabilizing co-solvents. Reformulation from phosphate buffer to succinate buffer increased adherence of the protein to aluminum hydroxide adjuvant from 15 to 91%, with a concomitant increase of nearly threefold in effective antigenicity in a mouse model. Using Fourier-transform infrared spectroscopy, we examined the secondary structure of the protein while it was adhered to aluminum hydroxide. Adjuvant adsorption produced only a small apparent change in secondary structure, while significantly stabilizing the protein to thermal denaturation. The vaccine therefore may be safely stored in the presence of adjuvant. Our results suggest that optimization of adherence of a protein antigen to aluminum adjuvant can be a useful route to increasing both stability and effectiveness, and support a role for a "depot effect" of adjuvant.

Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.

The influence of mutationally induced changes in protein folding on development of effective neutralizing antibodies during vaccination remains largely unexplored. In this study, we probed how mutational substitutions of streptococcal pyrogenic exotoxin A (SPEA), a model bacterial superantigen, affect native conformational stability and antigenicity. Stability changes for the toxin variants were determined using circular dichroism and fluorescence measurements, and scanning calorimetry. Self-association was assayed by dynamic light scattering. Inactivated SPEA proteins containing particular combinations of mutations elicited antibodies in HLA-DQ8 transgenic mice that neutralized SPEA superantigenicity in vitro, and protected animals from lethal toxin challenge. However, a highly destabilized cysteine-free mutant of SPEA did not provide effective immunity, nor did an irreversibly denatured version of an otherwise effective mutant protein. These results suggest that protein conformation plays a significant role in generating effective neutralizing antibodies to this toxin, and may be an important factor to consider in vaccine design.