Peter Burkhard - US grants

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5). This research project focuses on a new mechanical design, analysis and simulation framework for protein molecules. The fact that proteins are nano devices developed through evolution by nature suggests that the development of biomimetic artificial nano machines based on polypeptide chain building blocks is not only promising, but may also be the most practical approach to meet these challenges. This project continues the recent developments under an NSF Small Grant for Exploratory Research (SGER) and builds on a numerical modeling platform called 'PROTOFOLD' developed by the research team. The project will make use of mechanical analysis and design tools to accurately model protein molecules as mechanical devices, hence facilitating understanding of their function or ability to design and manipulate protein based molecular devices for specific functional purposes. The multi-disciplinary research team includes expertise in engineering, biology and pharmacology and will validate the proposed analytical tools and benchmark them in practical applications including nano-material, vaccine and drug design.

If successful, the proposed research will provide the ability to develop devices of increasing sophistication at the nano level, capable of acting as sensors, or carrying out more complex tasks. The broader impacts of this project range from proteomics, medicine, and rational drug design to biocomputers, bio robots, and biosensors. The interdisciplinary nature of this project will also have substantial educational impact on the students that will be supported through this program as well as on other graduate and undergraduate students through the proposed modules in mechanics of peptide based nano systems to be included in four separate courses. The outreach activities of this program include outreach to K-12 classrooms through the infrastructure already in place, and to the general public through the science museum in Hartford.

DESCRIPTION (provided by applicant): The development of a vaccine against P. falciparum has proven to be a difficult bioengineering challenge. Malaria afflicts 500 million people worldwide and annually kills about 3 million people, mostly children. "RTS,S", the most successful malaria vaccine candidate to date, provides only about 40% protective efficacy in humans in clinical and field challenge studies, and it requires formulation with an adjuvant to achieve protective immunogenicity. The application's broad, long-term objective is to develop self-assembling polypeptide nanoparticles (SAPN) as the basis for a P. falciparum malaria vaccine that is potentially more protective than those currently in clinical trials, shows excellent heat stability, and can be delivered without an adjuvant, which is likely to be consistent with formulations suited for delivery without a cold chain. SAPN assemble from linear polypeptide (LP) building blocks. Each LP consists of pentameric and trimeric coiled-coil domains separated by a linker, with epitope antigens displayed internally and on the N- and C- termini. The proposed work aims to identify the best configuration for the display of B cell and T cell epitopes on SAPN for producing the most potent immunogenic and protective immune response. SAPN displaying the P. berghei CSP epitope (analogous to the P. falciparum CSP B-cell epitope in RTS,S) have been shown to stimulate a long lasting protective immune response against a lethal sporozoite challenge in the P. berghei mouse malaria model without the need for an adjuvant. The SAPN particles have been shown themselves to have adjuvant activity. This suggests that the SAPN platform is potentially superior to other protein vaccine technologies used to date as these have all required the addition of extraneous adjuvants to be protective. The SAPN vaccines in our development pipeline will be evaluated for their mechanism of immune enhancement (adjuvanticity);protective efficacy against challenge with a strain of P. berghei (mouse malaria) expressing the CSP transgene from P. falciparum (human malaria);and immunogenic/protective response provided by specific P. falciparum T cell epitopes in human HLA backgrounds using human-HLA transgenic mice. The Specific Aims are to: (1) Design and produce SAPN in order to determine the optimal density of the immunodominant PfCSP B-cell epitope to stimulate a potent antibody response;(2) Determine the enhancement to the immune response provided by the addition of pan allelic HTL and CTL epitopes to SAPN with the optimized density of immunodominant PfCSP B cell epitope;and (3) Evaluate the safety, reactogenicity and immune response to the lead PfCSP SAPN vaccine candidate in a non-human primate (NHP) model. This R&D plan is expected to advance an innovative vaccine platform and to develop a malaria vaccine based on B cell and T cell epitope antigens from P. falciparum CSP that have already demonstrated successful, though limited, protective efficacy in clinical and field trials as RTS,S in AS02A. Success should qualify the proposed research for further funding for human- use product development and vaccine trials. PUBLIC HEALTH RELEVANCE: A malaria vaccine will be developed based on a new technology, self-assembling peptide nanoparticles, that display malaria parasite immunogens previously shown to elicit protective immunity to malaria. This new vaccine platform holds promise for ultimately producing an inexpensive malaria vaccine for the developing world.

DESCRIPTION (provided by applicant): We have recently developed a novel type of vaccine platform, so-called self-assembling polypeptide nanoparticles (SAPNs). We have demonstrated that these SAPNs are highly immunogenic and induce a strong B cell immune response characterized by high antibody titers without the need of adjuvant. We have engineered and biophysically characterized SAPNs to which activated nicotine hapten molecules can be coupled. Antibodies induced upon immunization with these SAPNs will bind nicotine in the blood stream and hence counteract the addiction-causing effect of nicotine. The overarching goal of the project is to demonstrate safety, tolerability and immunogenicity of the SAPN nicotine vaccine in a clinical trials phase I study. To achieve this goal we propose to first establish the immunogenicity of these nicotine-SAPNs in mice and to optimize their design with respect to the density of the hapten molecules, the SAPN concentration, and the route of immunization to obtain the highest possible antibody titer. Next, we will optimize the SAPNs for human use by engineering pan-DR binding epitopes that are specific for the human haplotypes into the SAPN scaffold. The immunogenicity of the SAPNs will then be verified in a second animal model followed by toxicity testings that will establish a safety profile of the SAPNs. The translation pah will then include protein formulation and lyophilization studies to optimize the long-term storage properties of the SAPNs. Along with the application for Swissmedic approval for clinical studies we will work on a large-scale production protocol of the final vaccine. The vaccine of the present project is adjuvant-free. This will eliminate all adjuvant induced side effects and reduce the pric as it will allow a simple galenic formulation of the vaccine. Since the SAPN-vaccines can be applied intranasally their patient acceptance is expected to be higher than for current vaccine approaches which require painful intramuscular

As indicated in the overview section of this PPG, vimentin consists of a central rod domain flanked by nona-helical head and tail domains. The central rod domain reveals a pronounced seven-residue periodicity, (abcdefg)n, in the distribution of apolar residues. Within this repeat, positions a and d are preferentially occupied by small apolar residues like Leu, lie. Met or Val, typical for a so-called coiled-coil structure (1). A coiled coil is formed by two or more a-helices wound around each other in a 'superhelix', and is a widespread structural motif in proteins (2, 3). This common structural motif enables IF proteins to self-assemble into 10-nm filaments in vitro In the absence of any auxiliary proteins or factors. These filaments are rope-like assemblies made from two to six 4.5-nm protofibrils (i.e., containing eight IF polypeptides each) which, in turn, are made of two intertwined 3-nm protofilaments each (4, 5). Although the molecular architecture of the 3-nm protofilament has not yet been precisely defined, it appears to be made from two antiparallel IF dimers, the latter being 2-stranded a-helical coiled-coils (i.e., formed via the central rod domain) of two parallel, in-register IF polypeptides (also see Figure 6).