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Rsv f protein compositions and methods for making same

a protein composition and protein technology, applied in the field of rsv f protein compositions and methods for making same, can solve the problems of purification, homogeneous, structure and refolding, and difficult to obtain immunogenic preparations

Inactive Publication Date: 2011-12-15
GLAXOSMITHKLINE BIOLOGICALS SA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0028]The invention relates to methods for preparing compositions and to compositions that contain RSV F protein, such as soluble RSV F ecto-domain polypeptides, including immunogenic compositions. The RSV F ecto-domain polypeptides can be in a single form, such as uncleaved monomers, uncleaved trimers, cleaved trimers, or rosettes of cleaved trimers. The RSV F ectodomain polypeptides can also be in two or more forms, for example two or more forms that exist in equilibrium, such as equilibrium between uncleaved monomers and uncleaved trimers. The invention provides several advantages. For example, the presence of a single desired form of RSV F in an immunogenic composition provides a more predictable immune response when the composition is administered to a subject, and more consistent stability and other physical and chemical characteristics when formulated into a vaccine.

Problems solved by technology

Due to the complexity of RSV F protein processing, structure and refolding, purified, homogeneous, immunogenic preparations are difficult to obtain.

Method used

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  • Rsv f protein compositions and methods for making same
  • Rsv f protein compositions and methods for making same
  • Rsv f protein compositions and methods for making same

Examples

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example 1

RSV F Polypeptides

[0466]This example provides sequences of a number of examples of polypeptides (e.g., that contain signal sequences) and nucleic acid sequences that may be used to express RSV F polypeptides of the present invention. The presented amino acid sequences include the signal peptide and contain an optional C-terminal linker and His tag (GGSAGSGHHHHHH (SEQ ID NO:90)). When these polypeptides are produced in host cells, the polypeptide will usually be processed by the cell to remove the signal peptide and, as described herein, some of the polypeptides will be cleaved, for example at unmodified furin cleavage sites. The invention includes compositions that contain, all forms of the particular RSV F protein ecto-domain polypeptides disclosed herein, including mature forms, which lack the signal peptide, forms that may be cleaved into subunits that comprise F1 and F2, and forms that lack the optional C-terminal His tag. The following examples are merely illustrative of the sc...

example 2

Expression and Purification of RSV F Constructs

[0481]The RSV F ECTO and truncated constructs, lacking the transmembrane domain and cytoplasmic tail region with either wild-type furin cleavage sites or harboring knock-out mutations to the furin cleavage sites and with or without prefusion stabilization mutations, were cloned into a pFastBac baculovirus expression vector (Invitrogen). Several of these constructs contain a C-terminal flexible linker followed by a His6-tag sequence used for chelating purification. The production of high-titer baculovirus stocks were passaged in Sf9 insect cells. Proteins were expressed by infecting either Sf9, Tn5 or High Five insect cells with the required baculovirus and harvesting the media supernatant two or three days post infection, monitored by western blot using an anti-RSV F or anti-6HIS antibody.

[0482]Large scale expression media was concentrated / purified by one of two general strategies for eliminating the deleterious Effect of the ferritin p...

example 3

Detection of Pre-Fusion and Post-Fusion RSV F

[0491]A number of methods are available to determine the conformation of the RSV F protein to assay whether a modification to the RSV F polypeptide or added molecule disfavors the post-fusion conformation. Examples include liposome association, conformation specific monoclonal antibodies (including as used in FACS, ELISA, etc.), electron microscopy, differential protease sensitivity between the conformations, gel filtration chromatography, analytical ultracentrifugation, dynamic light scattering, deuterium exchange NMR experiments, mass spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, tryptophan spectroscopy, and X-ray crystallography.

Liposome Association

[0492]Liposome association may be used to assay the conformation of the RSV F protein. Soluble forms of the RSV F protein in the pre-fusion conformation will not associate with liposomes while the post-fusion conformation will associate with liposomes.

[0493...

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Abstract

The present invention relates to immunogenic compositions comprising RSV F protein, methods for preparing compositions that contain RSV F protein ecto-domain polypeptides, and to certain engineered RSV F proteins and nucleic acids that encode the engineered RSV F proteins. Compositions prepared using the methods can contain RSV F protein ecto-domain polypeptides in a predominant or single desired form and conformation. The invention also relates to methods for inducing an immune response to RSV F.

Description

RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Patent Application No. 61 / 225,805, filed on Jul. 15, 2009, and U.S. Patent Application No. 61 / 294,426, filed on Jan. 12, 2010. The entire teachings of the above applications are incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]Respiratory syncytial virus (RSV) is an enveloped non-segmented negative-strand RNA virus in the family Paramyxoviridae, genus Pneumovirus. It is the most common cause of bronchiolitis and pneumonia among children in their first year of life. RSV also causes repeated infections including severe lower respiratory tract disease, which may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems.[0003]To infect a host cell, paramyxoviruses such as RSV, like other enveloped viruses such as influenza virus and HIV, require fusion of the viral membrane with a host cell's membrane. For RSV the conserved fusion protein (RSV F) fu...

Claims

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Application Information

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IPC IPC(8): A61K39/155C07H21/04A61P37/04C07K14/135
CPCA61K39/00C07K2317/76A61K2039/5258A61K2039/53A61K2039/55505A61K2039/55555A61K2039/55561A61K2039/55572C07K14/005C07K16/1027C07K2319/00C07K2319/21C07K2319/50C07K2319/73C12N2760/18522C12N2760/18534A61K2039/5252A61K2039/5256A61K2039/55566C12N2770/36143A61K39/155A61K39/12A61P11/00A61P31/14A61P37/04
Inventor SWANSON, KURTDORMITZER, PHILIP R.
Owner GLAXOSMITHKLINE BIOLOGICALS SA
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