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Over the last 200 years, the use of vaccines has proven to be one of the most successful medical interventions in the reduction of infectious disease. However, many challenges still remain with regard to fully realizing the health benefits of active immunization programs. Some of these obstacles include the implementation of improved adjuvants, development of single dose vaccines, methods to over come the poor immunogenicity of recombinant and subunit immunogens, and the ability to rapidly and rationally develop vaccines against emerging pathogens. <P>
In this regard, the overall objective of this R03 application is to define the adjuvanticity of novel polymer nanoparticles and the ability to induce protective immunity against the anthrax toxin using a single dose vaccine regimen employing these novel polyanhydride nanoparticles as the delivery vehicle. This objective will be achieved by using novel biodegradable polymer adjuvants with tailored chemistry to immunize mice with the recombinant protective antigen (rPA) of Bacillus anthracis. In addition, these studies will assess the immunogenic stability of the encapsulated rPA following extended storage (e.g., 18 months). <P>
The insights gained following the successful completion of these studies will provide the basis for designing immunomodulatory polymer adjuvants that will impact the development of stable, single-dose, non-toxic vaccines that will provide long term protection against other common diseases (e.g., diphtheria, tetanus), autoimmune diseases (e.g., multiple sclerosis) and/or allergic reactions. <P>
Several rationales for pursuing these studies are that single-dose vaccines are cost-effective, reduce the need for multiple injections, and result in greater patient compliance (i.e., no need for a return visit). In this regard, the implementation of vaccine delivery systems based on biodegradable polymers would offer significant therapeutic potential as these compounds will act as effective adjuvants and can be specifically tailored to provide release of antigen over time to maintain host immunity to the specific agent. However, the mechanism of adjuvanticity and the ability of manipulate adjuvant chemistry to selectively modulate the immune response is still largely unknown. <P>
Hypothesis: Encapsulation of rPA into polyanhydride nanoparticles will induce long-lasting, high titer protective antibody responses utilizing a single-dose immunization regimen.<P>
Specific Aims: <OL> <LI> To demonstrate the structural and antigenic stability of rPA encapsulated into polyanhydride nanospheres. <LI>To demonstrate the ability of novel polymer adjuvants to induce enhanced immune responses against the rPA of Bacillus anthracis.
NON-TECHNICAL SUMMARY: Even with impressive advances in medical care witnessed over the past century, infectious diseases remain the second leading cause of death worldwide accounting for 26% of all deaths. In addition, there are ever increasing public health concerns associated with the threat of bioterrorism. There is need to develop novel adjuvants that will allow for single dose administration and induction of protective immunity against the anthrax toxin. The overall objectives of this application are to demonstrate the immune enhancing capabilities of polymer adjuvants based on novel biodegradable polyanhydride chemistries to modulate the induction and activation of host immune responses against the recombinant protective antigen (rPA) of Bacillus anthracis. The specific aims of this project are: 1) to demonstrate the structural and antigenic stability of rPA following encapsulation into polyanhydride nanoparticles; 2) to demonstrate the ability of novel polymer adjuvants to induce enhanced immune responses against the rPA of Bacillus anthracis. Single-dose vaccines are cost-effective, eliminate the need for multiple injections, and result in greater patient compliance (i.e., no need for a return visit). Development and application of this adjuvant technology will facilitate the rational design of vaccines and the ability to appropriately redirect the immune response to develop protective immunity. The insights obtained during the performance of these studies will provide the basis for designing immunomodulatory polymer adjuvants that will impact the development of safe, stable, single-dose, non-toxic vaccines that may provide long term protection against other common diseases (e.g., diphtheria, tetanus, polio). All together, this integrated approach brings together expertise in polymer chemistry, immunology, and bacterial pathogenesis that will provide novel insights into the activity of polymer adjuvants as it relates to the relationships between adjuvant chemistry and induction of protective immunity.
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APPROACH: Specific Aim 1: To demonstrate the structural and antigenic stability of rPA encapsulated into polyanhydride microspheres. Two copolymer compositions have been chosen for these studies (20:80 and 50:50 CPH:SA). The stability of rPA will be assessed by incubation in saturated solutions of SA and CPH diacids. Protein samples will be incubated and gently agitated (100 rpm) for 1 week. Structural analyses of the protein samples will be performed in triplicate using SDS-PAGE, Far UV Circular Dichroism (CD) and fluorescence spectroscopy. The antigenic stability of rPA will be measured by ELISA. Nanoparticles of both 20:80 and 50:50 CPH:SA compositions will be prepared using the nano-S/O/O method. In order to evaluate the kinetics of protein release, aliquots of nanoparticles (15 mg) loaded with rPA will be placed in 1 mL of phosphate buffer and incubated at 37-C and shaken at 100 rpm. At different times, aliquots of supernatant will be collected. In order to identify the formulation of rPA-containing nanoparticles that induces the most rapid rise in anti-PA antibodies and the formulation that gives the highest and most long-lasting titers, two formulations will be tested with 3 doses of rPA. 12 mice per group will be vaccinated. From each mouse, serum samples will be collected via the saphenous vein and tested for rPA titers on various days post-immunization. Because induction of effective CD4 T cell help has been shown to be critical for maintaining antibody titers, antigen-specific recall responses of CD4 T cells will be assessed at 40 and 80 days post-immunization. Six mice in each group will be euthanized at each of the two time points and the lymph nodes (LNs) draining the site of immunization (e.g., brachial and axillary LNs) will be aseptically excised. LNs will be dissociated into single cell suspensions, the cells labeled with CFSE and inoculated into 96 well culture dishes. The cell cultures will be stimulated with anti-CD3/CD28 (positive control) and graded doses of rPA (0.1, 1.0, and 10 ug/mL). The nanoparticle vaccine will be compared to 1 and 10 ug of rPA administered in alum as part of the standard prime-boost regimen. 12 mice per group will be vaccinated. All mice will be bled for rPA titers . Serum antibody responses and CD4 T cell responses will be evaluated as indicated above. We anticipate that these dose response experiments will allow us to identify the immunization regimen (i.e., nanoparticle formulation and rPA dose) that gives the most rapid rise in anti-PA titers (post-exposure formulation) and the highest and most long-lasting titers (pre-exposure formulation).