Title

Self-assembling Polypeptide Nanoparticles: Design, Synthesis, Biophysical Characterization and Biomedical Applications

Date of Completion

January 2010

Keywords

Chemistry, Biochemistry|Nanotechnology|Biophysics, General

Degree

Ph.D.

Abstract

Inspired by the architecture of icosahedral viruses, self-assembling polypeptide nanoparticles (SAPN) with icosahedral symmetry were developed. The building block for the SAPN was a single polypeptide chain. Similarly, the capsid of quite a few small viruses are built from one single peptide chain. The polypeptide chain of the SAPN consists of a pentameric coiled-coil domain at the N-terminus joined by a short linker segment to a trimeric coiled-coil domain at the C-terminus. ^ Here we have studied factors governing self-assembly of the SAPN such as linker constitution and trimer length. The interdomain linker 2i88 afforded the most homogenous nanoparticles as verified by TEM and DLS. Furthermore, AUC and STEM analyses suggest that the nanoparticles formed using the linker 2i88 have a T=3-like architecture confirming computer modeling predictions. As for trimer length, we have shown that it is possible to synthesize SAPN with a trimer that is as short as only 17 amino acids. ^ Given that the N-terminus and C-terminus of the SAPN can be extended to include epitopes and give rise to a repetitive antigen display system, vaccine applications of the SAPN were also investigated here. We grafted parts of the SARS virus' spike protein onto our SAPN to repetitively display this B-cell epitope. Biophysical characterization showed that single nanoparticles of the expected size range were formed. Immunization experiments in mice at University of Colorado Denver revealed that the antibodies elicited were conformation-specific. Moreover, the antibodies significantly inhibited SARS virus infection of Vero E6 cells. ^ SAPN were also functionalized at the C-terminus with a B-cell epitope from the circumsporozoite protein (CSP) of the malaria parasite Plasmodium falciparum and at the N-terminus with CTL epitopes from CSP. The trimeric coiled-coil domains of these malaria SAPN were modified to include a HTL epitope. Even will all these modifications, self-assembly occurred as confirmed by TEM and DLS. In immunization experiments performed at WRAIR good immune responses were obtained. ^ Another biomedical application of SAPN is the development of a peptide-based serodiagnostic assay for tuberculosis (Tb). In an ELISA format, Tb-SAPN showed modest responses in serodiagnosis of Tb. ^