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In addition, tissue distribution experiments indicate that the lipid-CDV conjugates are not deposited in the kidney, suggesting the possibility of diminished nephrotoxicity.


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Specific aims and milestones that represent critical activities and key Studies 19 decisions in this proposal are: 1. Synthesize and characterize adequate drug substance to complete Aims 2 through 4. Characterization will include preformulation studies. Alternative routes of synthesis will also be examined.

The data generated in aims 1 through 4 will be used to choose which candidate to carry into full development first milestone. Complete absorption, distribution, metabolism and elimination studies necessary to file an IND. Under the animal efficacy rule Federal Register , , this study could provide the efficacy data necessary for FDA approval.

Manufacture prototype formulations, and produce cGMP clinical trials material. Upon FDA approval, a Phase I trial will be initiated to evaluate the safety, tolerability and pharmacokinetics of a single, escalating dose in human volunteers. Because smallpox was eliminated from the U. The subsequent 40 years have produced a population that is immunologically naive and highly susceptible to orthopoxvirus infection.

Due to the small but significant risk of serious complications from vaccination, mass immunization of the populace is contra-indicated. The target of our antiviral drug development efforts will be the poxvirus proteinase responsible for core protein maturation, a step which is absolutely essential for the production and spread of infectious virions.

This project will be carried out as a partnership between an academic group at Oregon State University with a long history of research in various aspects of poxvirus proteolysis, and a biopharmaceutical company, SIGA Research Laboratories, which is actively engaged in the development of proteinase inhibitors as anti- infectives. Together, these groups will identify' the viral gene product responsible for catalyzing core protein maturation and use genetic approaches to validate it as an antiviral target.

Expression vector technology will be used to express and purify the large quantities of the core protein proteinase. The purified proteinase will serve as the starting material for a two-pronged approach to the identification of potential inhibitors: l Structure-function analysis coupled with rational drug design; and 2 Development of an in vitro cleavage assay appropriate for use in high-throughput screening against limited libraries of potential proteinase inhibitors.

If necessary, lead compounds will be subjected to iterative chemistry to improve bioavailability, specificity and potency. The most promising optimized lead compound s will then be selected and advanced into preclinical and toxicology studies in preparation for in vivo testing in a murine and1or primate challenge in collaboration with NIAID and USAMRIID investigators. It is anticipated that the results of these experiments will identify one or more antiviral drugs as development candidates to provide a rapidresponse defense against the deliberate introduction of a pathogenic poxvirus into the environment.

An event which we all hope never transpires, but for which preparation is vital. However, the vaccine has known complications, especially in immunocompromised hosts, pregnant women and infants. In past vaccination efforts, such complications were treated in the U. Although this material had efficacy, little is known about which components of the immune globulin were effective and there could be batch-to-batch variation in efficacy.

Moreover, since routine vaccination has been discontinued for many years, there is only a limited supply of VIG available, and there are concerns about its safety. Our long-term goal is to develop a cocktail of defined and high affinity antibodies to VV proteins that will replace the use of traditional VIG in the event that mass VV vaccination is needed.

A cocktail of such antibodies, which we will call VIG-R replacement will provide a uniform and secure source of a VV immune therapeutic reagent.

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In this application, we propose experiments in four specific aims. These are: 1 to express and characterize VV envelope proteins LIR, A33R and B5R in a baculovirus expression system and to prepare mouse and rabbit antibodies to those proteins; 2 to develop and characterize human monoclonal antibodies to LIR, A33R and BSR produced using phage display; and 3 to test the ability of immune reagents against VV proteins to protect mice from challenge with VV.

Fulfillment of the aims of this grant should provide new information about each of the VV proteins and antibodies, as well as a source of reagents that can comprise a VIG-R. If our approach appears promising, we will expand this study to include other VV proteins. The reagents developed in this grant application will also be valuable for basic studies concerning the role of these proteins in the VV life cycle and could themselves be considered as vaccine candidates in future investigations.

It will function as an integral component of the Region VI RCE's mission by opening up new lines of research and relevant product development. The plan takes advantage of the region's wealth of relevant scientific expertise to allow for maximal utilization of Developmental Research as a tool to explore promising research leads. The scientific leadership for research projects will be provided both by investigators who are already established in biodefense research, as well as others willing to apply their expertise to a novel aspect of product-oriented biodefense research.

In this way, the Developmental Research program will complement the Career Development program in increasing the numbers of dedicated biodefense investigators. An essential feature of this plan is a systematic and rigorous management approach that will allow for the selection of the most relevant and scientifically sound research projects and that will allow for an effective monitoring and evaluation process. The Program will be managed by an Associate Director of the RCE and will draw upon the expertise of the RCE Scientific Advisory Board to reach decisions concerning continued support of productive developmental research versus early discontinuation of unproductive projects, and selection of new projects for funding under this program, thereby contributing to the establishment of a product development pipeline within the RCE.

To respond to this specific threat, the United States must have at its disposal supplies of both vaccinia virus vaccine and antiviral compounds directed against smallpox infection. The antiviral compounds are needed in situations in which the vaccine is contraindicated, such as immunosuppression. In addition, the antiviral compounds are needed to blunt adverse complications that are known to be associated with vaccine administration.

An ideal antiviral should also be able to directly prevent smallpox infection in situations where vaccine delivery is delayed. A strongly favorable feature of PFs is that they function with their own POLs, so that an antiviral that targets a viral PF should be very specific and not interfere with cellular replication. A newly developed mechanistic rapid plate assay will be used to screen thousands of potentially inhibitory 22 Smallpox compounds.


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This high throughput screening will be followed by procedures to evaluate the inhibitory compounds and to ultimately test their ability to block vaccinia virus infection. A drug that prevents vaccinia processive DNA synthesis will be useful in curtailing vaccinia vaccine complications. The approach may help deliver the 'just-in-time' need for reagents to combat a smallpox bioterrorism threat.

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Eradication of smallpox as a biothreat is now our objective. New stockpiles of safe and efficacious smallpox vaccine are needed to protect both the civilian and military populations against deliberate release of the smallpox virus. Currently there is no commercially available vaccine, and the previously approved one is a live vaccinia inoculum associated with more adverse events than any other approved vaccine.

Recent work has focused on improving the manufacturing process of the original vaccinia vaccine strain and testing other live attenuated viruses. We propose to discover new vaccine candidates from viral components.


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A subunit design would be safer and more easily controlled during manufacture. Since we anticipate obtaining multiple protective subunits, these can be mixed and matched to effectively defend against wild type, natural variants, and bioengineered smallpox isolates. To identify antigens of smallpox that carry vaccine potential, the goal of our proposed project is to screen all the genes of the closely related cowpox virus for their ability to protect against disease in its natural murine host.

This genome-level approach is feasible because the viral genome databases are available and we have developed the platform technologies that make a comprehensive screen fast and efficient. We will establish the electronic and molecular protocols to synthetically generate a thousand codon-optimized gene sequences. It will be used to produce a library of high quality cowpox subgenes. An advanced library screening method employing multiplex arrays will enable us to screen all the subgenes for protection in one experiment.

Vaccine candidates will be confirmed and immune characterized. Both the cowpox candidates and their variola homologs will be formatted three ways and evaluated in immune and cowpox protection assays. This project will uncover new subunit vaccine candidates against variola and prepare them for final validation in a primate challenge experiment. Polynucleotide vaccines are on the forefront of Studies 23 vaccine development.

They are important because of the fast development times possible and because cell mediated immune responses can be induced. The delivery system proposed here will be effective for most polynucleotide vaccines. In addition to Biodefense, this system will provide effective polynucleotide vaccine delivery for less lethal viruses, some cancers and some third world diseases.

The defense and commercial applications are extensive. The polynucleotide vaccine delivery system described here uses a microneedle array with the polynucleotide coated right on the needle in the array. There are hundreds of needles each about 0. This array in inserted into the skin with the needle penetrating to about the basal lamina. After insertion the polynucleotide leaves the needle surface and an electric field is used to permeabilize dendritic and epithelial cell membranes to permit the polynucleotide to enter the cell.

Arteriviruses

The specific aims of this project are to design and develop to FDA QSR Standards the vaccine delivery system prototype and to test the prototype in a human trial. This is a fast-track application. In Phase I, a system design will be completed including the hand-piece, microneedle array and miniature waveform generator. The coating chemistry and specific waveforms will be optimized in mice.

In Phase II, a prototype of the final design will be completed. Safety and efficacy will be demonstrated in mice and safety will be demonstrated in humans. Little is known about 1 the antigens and epitopes targeted by the cellular responses in humans immunized with vaccinia virus, and 2 which responses are crossreactive with variola virus and hence would be expected to contribute to the protection engendered by the vaccine.

In the first part of the studies proposed herein, we will 1 determine immunodominant antigens recognized by Class I and Class II restricted responses in humans immunized with vaccinia virus, 2 map the epitopes recognized within each antigen, and 3 determine their degree of crossreactivity with homologous variola virus-derived sequences.

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We anticipate that these studies will lead to the definition of a broad range of epitopes, facilitate a rigorous definition of correlates of protection against smallpox infection in humans, and also enable the evaluation of the performance of different vaccine candidates. The vaccinia-based vaccines currently available, while effective, are associated with significant and serious, albeit rare, side effects. Because of these side effects, and because of the worldwide eradication of variola virus, vaccination of the general population was deemed as no longer 24 Smallpox desirable. Recent renewed concerns have been raised over bioterrorist use of the virus.

In the context of the studies proposed herein, a concern could be raised that if the vaccinia-induced protection is mediated by relatively few immunodominant and crossreactive antigens, a modified smallpox virus could be engineered that lacks those crossreactive epitopes. Under this terrifying scenario, the protection elicited by the vaccinia would be ineffective against the biological weapon. In the second part of the grant, we propose to counter this risk through the identification of variola virus-specific determinants derived from immunodominant antigens in the context of the vaccinia virus responses, but not crossreactive with the homologous variola virus sequences.

These variola virus-derived epitopes would be incorporated in an optimized multideterminant vaccine construct, inserted in the currently available vaccinia vaccine. It also has well established utility in disease therapy e. Transgenic mouse models of Alzheimer's disease AD develop high density A-beta deposits in cerebral cortex and hippocampus, neuritic changes and, ultimately, inflammatory reactions to these deposits.

Unfortunately, the functional consequences of this treatment could not be effectively assessed in these mice, owing to severe learning and memory deficiencies observed early in the lifespan. These mice develop learning and memory deficits which correlate with the accumulation of A-beta, deposits. They predict different outcomes depending on the age of vaccination.