Fusion proteins of ciliate granule lattice proteins, granular protein particles thereof, and uses therefor
Inventors
Clark, Theodore G. • Bisharyan, Yelena • Papoyan, Ashot • Anderson, Kyle • Colussi, Paul
Assignees
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Publication Number
US-9512181-B2
Publication Date
2016-12-06
Expiration Date
Abstract
This invention is directed to methods for the production of immunogenic granular particles. In certain embodiments, the invention is directed to methods and products for the production of immunogenic granular particles produced in ciliates. In certain embodiments, the invention is directed to compositions comprising Granule lattice protein/Antigen(Grl/Ag) fusion polypeptides.
Core Innovation
The invention relates to an immunogenic composition comprising self-assembled granular particles of a fusion polypeptide. The fusion polypeptide comprises a granule lattice protein (Grl) moiety and an antigen (Ag) moiety, where the Grl moiety is selected from Grl1 to Grl9 or has at least about 95% amino acid sequence identity to specified Grl sequences. The particles self assemble into granules in a medium comprising a divalent cation.
The granular particle self assembly is driven by the presence of a divalent cation in the medium at a concentration of at least about 0.01 mM, and the claims also recite a higher threshold of at least about 0.5 mM. The platform includes granular particles that present heterologous antigen moieties in immunogenic granular form and can include single or multiple different Grl/Ag fusions.
The disclosure positions the self-assembled Grl/Ag granular particles to induce potent humoral and cellular immune responses. The document also describes an antigen scope spanning multiple antigens, including influenza hemagglutinin and other listed antigen classes, and references particle formation under divalent cation exposure and acidic pH.
Claims Coverage
The independent claim covers an immunogenic composition defined by self-assembled granular particles formed from a Grl/Ag fusion polypeptide under divalent-cation conditions, with the Grl moiety restricted to Grl1 to Grl9 or specified sequence identity. Dependent claims further refine the divalent-cation concentration threshold, specify particular divalent cations, constrain particle characteristics, and broaden antigen scope and composition features.
Self-assembled granular particles of a Grl/Ag fusion polypeptide
An immunogenic composition comprising self-assembled granular particles of a fusion polypeptide comprising a granule lattice protein (Grl) moiety and an antigen (Ag) moiety, wherein the particles self assembled into granules in a medium comprising a divalent cation.
Grl moiety defined by Grl1 to Grl9 selection or specified sequence identity
The Grl moiety is selected from Grl1 to Grl9; or the Grl moiety has at least about 95% amino acid sequence identity to SEQ ID NO: 1 through SEQ ID NO: 9.
Divalent cation-mediated self-assembly threshold concentration
The particles self assembled into granules in a medium comprising a divalent cation when said divalent cation was present in the medium at a concentration of at least about 0.01 mM.
Raised divalent-cation concentration threshold for self-assembly
The particles self-assemble in a medium containing a divalent cation at a concentration of at least about 0.5 mM.
Specified divalent cation Ca2+
The divalent cation is Ca2+.
Particle mean diameter range
The particles have a mean diameter in the range of about 20 nm to about 200 nm.
Antigen moiety defined by pathogen-class polypeptides
The antigen moiety is one of the specified pathogen, bacterial, viral, protozoan, or worm polypeptides.
Mixture of two or more different Grl/Ag fusion polypeptides
The immunogenic composition includes two or more different Grl/Ag fusion polypeptides.
Overall, the claims cover immunogenic compositions formed by self-assembled granular particles of a Grl/Ag fusion polypeptide, where the Grl moiety is limited to Grl1 to Grl9 or near-identical sequences and self assembly occurs in the presence of divalent cations above specified concentration thresholds. Dependent claims further refine divalent cation selection, particle size, antigen scope by pathogen class, and compositions containing multiple different Grl/Ag fusions.
Stated Advantages
Induces potent humoral and cellular immune responses.
Provides improved mouse survival after influenza challenge for HA-Grl1 granular formulations.
Documented Applications
Influenza-related immunogenic formulations using HA-Grl1 (H5N1) that form immunogenic granular particles and are associated with improved mouse survival after influenza challenge.
Use of Grl/Ag fusion constructs to form immunogenic granular particles upon exposure to divalent cations and acidic pH.
Examples employing pfs48/45-Grl1 and flagellin-Grl4 constructs to support particle formation or observations.
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