Robert J. Collier - US grants

A major limitation in the toxicity of chimeric toxins is the efficiency with which enzymically active portions of toxin moieties are transferred into the cytosolic compartment of susceptible cells. Our primary goals are to elucidate features of diphtheria and Pseudomonas toxins that function in the transfer and to build a technological base for using molecular genetics to alter these toxin moieties in desired manners. We have recently cloned, into E. coli, a diphtheria toxin gene fragment that encodes the toxin lesder peptide, the A fragment, and the hydrophobic portion of B and have also obtained a cloned form of the Pseudomonas toxin gene. These are now being subjected to site-directed mutagenesis in order to probe structure/function relationships in the two toxins, including catalytic activities and membrane transport functions. Concurrent studies on the protein level, including lipid bilayer studies and binding and transport studies in tissue culture, are being undertaken in order to provide a basis for understanding in greater detail the limiting factors in the transfer of enzymically active portions of toxin moieties into the cytosolic compartment. (HI)

X ray crystallography; laboratory mouse; site directed mutagenesis; transposon /insertion element

Generating immunogenic, nontoxic forms of bacterial toxins (toxoids) is a potentially important application of recombinant DNA technology. The current project has two major goals: (1) to generate an effective recombinant toxoid of Pseudomonas aeruginosa exotoxin A (ETA), and (2) to provide a model system for generating toxoids by mutagenizing active- site residues for cloned toxins. We have used photoaffinity labelling to identify glutamic acid-553 (Glu-553, or E553) as a crucial active- site residue of ETA and employed oligonucleotide-directed mutagenesis to delete this residue or replace it with other amino acids. In continuing these studies we will focus on ETA-E553X, a candidate toxoid formed by specifically deleting Glu-553. We will develop improved methods for producing ETA-E553X and other cloned recombinant antigens in quantity in P. aeruginosa, and perform a more thorough characterization of the biological and biochemical properties of ETA-E553X. This mutant toxin will be subjected to phase 1 safety and immunogenicity trials in humans, and will be used to prepare protein-polysaccharide conjugates with various P. aeruginosa polysaccharide components. We will also generate and characterize other candidate recombinant toxoids of ETA. These studies may have important applications in controlling P. aeruginosa infections; and lessons learned about toxoid construction by mutagenizing active-site residues could be widely applicable.

Biochemical instrumentation will be acquired for use in research projects in a broad range of basic biological disciplines, including developmental biology, microbiology, protein chemistry, cell biology, biochemistry, cell physiology, molecular biology, and biotechnology. The equipment, which includes microchemical instrumentation for DNA, RNA, and protein chemistry, and ultracentrifuges and related equipment for basic biochemical manipulations, will be part of a shared facility. Access to modern equipment is critical for progress in contemporary biology and in biotechnology-related research.

It has been shown by several investigators that fetal retinal tissue can be successfully transplanted to various cortical locations or into the eyes of normal and lesioned rats. Morphological assessment of such transplants have an appearance resembling the normal retina. Although electrical responses have been recorded from retinal transplants placed in cortical locations, it is not known if retinas transplanted into the eye are capable of eliciting a response to photic stimulation or if these transplants are capable of transmitting that response to the host retina and ultimately to higher cortical areas. The experiments described in this proposal have been designed to determine the functional limitations of transplantation of retinal tissue into the eyes of light-blinded host rats. Physiological functioning of the transplants will be assessed by electroretinography and visually evoked responses. The findings of the proposed experiment will provide a basis for determining the feasibility of using retinal grafts to restore retinal function. In addition, this pilot data will serve to introduce this newly established investigator to a new an important aspect of visual science and would be the basis for future support.

Although most HIV vaccine approaches investigated in the past have focused on generating neutralizing antibodies against the virus, there is increasing evidence to suggest that the key to success of this endeavor lies in strategies for eliciting cellular antiviral immunity. The applicants have proposed experiments aimed at developing a peptide vaccine with the capacity to prime a cytotoxic T- lymphocyte (CTL) response in vivo aginst HIV-1 epitomes. The nef and gag proteins have been identified as the sites of potentially important epitomes and will be the primary focus of their work. In order to prime a CTL response, a CTL epitome-containing protein must be delivered to the cytosol of mammalian cells. There, it is processed to form peptides which are then complexed with MH class I molecules and presented at the cell surface. Recent studies have revealed how the intra cellularly acting toxin, such as the anthrax toxin (AT), may be modified to ablate its toxicity and enable it to deliver heterologous proteins to the mammalian cell cytosol. As a ramification of these findings, the applicants have demonstrated that AT fusion proteins containing selected epitomes from Listeria monocytogenes and lymphocytic choriomenigitis virus may be used to prime protective CTL responses in mice against these pathogens. In the research proposed, the applicants intend to prepare similar AT fusions continuing the nef and gag proteins and test these constructs for ability to prime an antigen-specific CTL response in mice. Furthermore, the applicants will also attempt to determine whether human cells treated invitro with these fusions can induce proliferation of gag- and nef-specific CTL from peripheral blood ofpatients infected with HIV. Finally, the researchers will investigate whether patient-derived gag- and nef-specific CTL can lyse haplotype-matched target cells treated with the fusion proteins. These experiments will allow the utility of the AT-based vaccine strategy to generate a CTL response against HIV antigens to be evaluated and, if positive indications are obtained, will set the stage for safety and efficacy studies of AT-based vaccines in primate animal models.

Microbes and their products have important uses in forensics. Diatoms have been used to identify where drowning victims went into the water and PCR tests have established paternity and relationships between crime victims and alleged perpetrators. New technologies, like PCR and genome sequencing, could be used to establish the origin of an infectious disease outbreak, whether natural or due to intentional human intervention (bioterrorism). Questions of how to interpret molecular data should not wait until case law determines their validity. For any new technology to be accepted, it is first important to determine what standards should be applied. Knowledge of the genome sequences of Bacillis anthracis (the cause of anthrax) and other pathogens on the Center for Disease Control and Prevention (CDC) list of potential bioterrorism agents must be augmented by knowledge of how this information can best be used. While there are well-established criteria for evaluating and interpreting the results of such classical methods as cultivation or serological testing, no similar criteria have been developed for the new molecular methods. This colloquium will 1) consider the use of microbial forensics in responses to disease outbreaks (including bioterrorist attacks on human populations or agriculture); 2) assess when and under what conditions new molecular techniques might be appropriate and whether they could be used in conjunction with more traditional approaches; 3) determine how genome sequences and other molecular data can be best used and interpreted; 4) suggest which standards can be defined and which cannot; and 5) identify potential problems and areas that need to be addressed experimentally so that criteria may be defined.

The American Academy of Microbiology, the honorific leadership group of the American Society for Microbiology, will convene a colloquium June 6-9, 2002, in Burlington, Vermont. The colloquium will bring together 25-30 interdisciplinary, expert scientists representing evolutionary microbiology, ecology, genomics, genetics, bioinformatics, forensics, chemistry, and clinical microbiology for structured deliberation on issues in microbial forensics. Participants will consider several theoretical scenarios and survey current molecular technologies, including genome sequencing, with a view to recommending standards for evaluation and validation of the data these technologies produce. The colloquium will produce the intellectual material for a published report with unbiased statements of the issues and practical recommendations for the future.

Animal Model; Animal Models and Related Studies; Anthrax; Anthrax Attack; Anthrax disease; Assay; B. anthracis; Bacillus anthracis; Bioassay; Biologic Assays; Biological Assay; Biological Terrorism; Bioterrorism; CAPS; Capsules; Cessation of life; Death; Glutamic Acid; High Throughput Assay; Immunization; Immunologic Stimulation; Immunological Stimulation; Immunostimulation; Infection; Injection of therapeutic agent; Injections; L-Glutamic Acid; Laboratories; Lead; Mammals, Mice; Mammals, Rodents; Mice; Murine; Mus; Pathogenicity Factors; Pb element; Rodent; Rodentia; Rodentias; Sensitization, Immunologic; Sensitization, Immunological; Testing; Therapeutic Intervention; Therapy, Vaccine; Toxic effect; Toxicities; Toxin; VAC-TX; Vaccine Therapy; Virulence Factors; anthracis; anthrax toxin; base; capsule (pharmacologic); experience; falls; heavy metal Pb; heavy metal lead; high throughput screening; inhibitor; inhibitor/antagonist; intervention therapy; macrophage; model organism; molecular mass; new therapeutics; next generation therapeutics; non-human primate; nonhuman primate; novel therapeutics