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Bacillus anthracis is a facultative intracellular bacterial pathogen that can cause cutaneous, gastrointestinal or respiratory disease in many vertebrates, including humans. Commercially available anthrax vaccines for immunization of humans are of limited duration and do not protect against the respiratory form of the disease. Brucella abortus is a facultative intracellular bacterium that causes chronic infection in animals and humans. As with other intracellular pathogens, cell mediated immune responses (CMI) are crucial in affording protection against brucellosis. B. abortus strain RB51 has been shown to be useful in eliciting protective cell mediated immunity and humoral responses against Brucella in cattle and other animal species. Since the protective antigen (PA) of B. anthracis is known to induce protective antibodies, it was decided that the objective of this research was to test whether the gene encoding PA could be expressed in Brucella producing a bivalent vaccine to protect against both brucellosis and anthrax. The pag gene was transcriptionally fused to promoters of genes encoding superoxide dismutase or heat shock protein groE, subcloned into a broad host range plasmid (pBBR1MCS) and shown to express in E. coli by immunoblotting using antiserum specific for PA. The immunoblot results revealed that E. coli produced a PA protein of the expected size. In addition, the culture medium was shown to contain the same PA protein using immunoblotting. These results show that E. coli is capable of expressing B. anthracis PA in both the cellular and extracellular forms. The pBB/PA plasmid was used to transform B. abortus RB51 and CmR clones screened for the expression of PA by immunoblotting. Twenty clones of strain RB51/pBBSOD were show to express a 30kDa PA protein. Three clones of strain RB51/pBBGroE-PA were shown to express a 63-83kDa protein as detected by antiserum specific for PA. Using the A/J mouse, an immunocompromised vertebrate model, immunization and challenge studies were performed. Preliminary results demonstrate that the bivalent vaccine is capable of producing protection against a live challenge with B. abortus and some protection against live non-disease producing spores of B. anthracis. / Master of Science

Bacillus anthracis has been classified as one of the most dangerous bioterrorism agents causing high mortality rates in short periods of time. Anthrax spores are extremely resistant to chemical and environmental factors, and have the ability to return into a vegetative (virulent) state during the process of germination. Previous research has suggested that spores can be eradicated with common disinfectants after germination and release of spore coats. During germination, the spore coat is degraded, making the spore susceptible to penetration of chemicals into the spore core. While previous research has focused on a qualitative understanding of germination of spores by obtaining high-resolutions images of spore coats to understand how protein coat layers change during germination, very few studies have evaluated changes in mechanical properties of spores during germination, and how germination affects virulence of macrophages. In this study, we performed a series of in vitro experiments to do an in-depth analysis of germination and virulence of B. anthracis. Atomic force microscopy (AFM) was used to investigate changes in spore surface properties during germination including morphology, roughness, elasticity, and spring constant. AFM results suggested that germination mechanisms depend on germinants used to trigger germination and roughness of Bacillus species increase during germination. In addition, the elasticity and spring cell constant of B. anthracis spores are affected during germination since the elastic moduli and cell spring constant values decreased with time as the spore was germinating, making the cells more susceptible. Spore killing was also tested both in sporulated and vegetative B. anthracis using the antimicrobial peptide chrysophsin-3 and the surfactant dodecylamine (DDA). Both killing agents were capable of eradicating B. anthracis spores, but more killing was observed for spores that were germinating or had become vegetative. The presence of germinant receptors from the Ger operon and its role on germination kinetics of B. anthracis was also investigated. The germination of mutant spores that carried one receptor or lacked all germinant receptors was compared to the germination kinetics of wild-type B. anthracis. Our results suggest that germination of spores is modified by the presence or absence of germinant receptors. Furthermore, the mutant B. anthracis strain lacking all receptors germinated suggesting that other receptor independent pathways may exist in B. anthracis. Finally the ability of B. anthracis to adhere, grow, and invade macrophages was investigated. Invasion of macrophages by B. anthracis was dependent on germinant receptors and the ability of spores to germinate and multiply. Our results suggest that macrophages were not capable of killing infecting spores, and on the contrary, germination of spores inside macrophages caused the lysis of macrophages. An uncontrolled release of cytokines by macrophages was elicited by spores and germinated B. anthracis. Our study helps understand the process of germination of B. anthracis spores at a nanomolecular level. Our investigation may be a valuable tool in the design and development of antisporal compounds.

Recombinant antibodies have increased in prominence as therapeutics and diagnostic tools since their introduction to the market in the mid-1980s. They are used to treat diverse conditions from Crohn's disease to cancer. Since the Anthrax letter attacks of 2001, a great deal of work has been carried out to develop therapeutics to this disease, and antibodies that neutralize the toxic action of Bacillus anthracis are prominent among them. This dissertation describes the elucidation of the structure of the 14B7 family of neutralizing antibodies directed at protective antigen (PA) of B. anthracis and the complex of PA domain 4 (PAD4) with an ultra-high affinity neutralizing antibody (M18), and then utilizes this information to aid in the engineering of the antibody to various ends. Chapter 2 presents the structure of the M18-PAD4 complex and of the 14B7 family of antibodies, which aids in the understanding of the affinity maturation process for this antibody family. Chapter 3 describes the affinity maturation of M18 to a PA variant by applying the knowledge gained from the complex structure. This previously intractable challenge was met by employing saturation mutagenesis in highly focused libraries to M18 directed by the complex structure to the area of variation on PA. These results indicate that this could be a generalizable method for the engineering of M18 to natural and deliberate variation of PA. Chapter 4 reports work toward the development of a reversible, photoresponsive antibody using small molecule and polymer-protein conjugates. The results indicate that a probable site on M18 was located for placement of the polymer appendage, although further work is necessary to empirically refine the properties of the photoresponsive polymer. Chapter 5 presents an unrelated project, which was to confirm the existence of a proposed RNA thermosensor in the 5' untranslated region of LcrF from the pathogenic bacterium Yersinia pestis, the causative agent of plague. Overall, these studies reveal the power of structure-based engineering in this antibody-antigen system. In addition, the structural elucidation of the M18-PAD4 complex and the 14B7 family of antibodies furthers our basic understanding of protein-protein interactions and the process of affinity maturation of antibodies. / text

Thesis (Ph. D.)--University of Nevada, Reno, 2008. / Includes bibliographical references. Online version available on the World Wide Web.

Inaug.-diss.-Bern. / Lebenslauf.

Thesis (M.S.)--Brigham Young University. Dept. of Chemical Engineering, 2005. / Includes bibliographical references (p. 101-110).

No description available.

Affinity maturation increases the affinity of B-cell derived antibodies to their cognate antigens. In this study, we characterized the kinetic, structural, dynamic and thermodynamic evolution of antibodies during affinity maturation. Through single B-cell cell sorting, paired heavy and light chain sequencing, phylogenetic analysis, antibody expression, and physicochemical characterization, we were able to longitudinally analyze the stages of affinity maturation of anti-PA (B.anthracis protective antigen) antibodies. Following repeated immunizations, we observed up to an 10,000-fold increase in antibody affinity, mainly through a decrease in the off-rates. For detailed maturation analysis, we chose three antibodies lying along a single clonal branch--the clone’s unmutated common ancestor (UCA), a medium affinity antibody (MAAb) appearing after second immunization, and a high-affinity antibody (HAAb) appearing after third immunization. Most of the mutations that occur between the UCA and HAAb resulted in key changes to structural conformation. In particular, mutations change residues in the CDR-H3 region inducing the folding of the CDR-loops into a conformation that is more complementary to PA. This advantageous new antibody conformation is preserved in the unbound state, indicating that though the UCA and MAAb appear to use an induced fit and/or conformational selection mechanism, the HAAb is more rigidly lock-and-key. Thermodynamic results support this interpretation. In the first maturation step from UCA to MAAb, enthalpic improvement indicates optimization of noncovalent interactions. The second step from MAAb to HAAb predominantly involves entropic improvement by which the advantageous conformation made accessible in the first step is made more dominant via the narrowing of effectively accessible conformations, which allows better contact with PA. This is also reflected by a less significant improvement in the enthalpic component of PA-binding. Studies examining the evolving protein-dynamic characteristics further support this interpretation. In summary, we observed that a single energetic component is not responsible for increased affinity in the maturation pathways we studied. From UCA to MAAb, affinity increases through optimization of noncovalent interactions. From MAAb to HAAb, affinity increase is achieved through changes that stabilize the favorable conformation in the unbound state. A better understanding of affinity maturation can have implications for antibody engineering and vaccine development.

No description available.