Virus like particles comprising target proteins fused to plant viral coat proteins

A capsid protein and plant virus technology, applied in the direction of viruses, viral peptides, drug combinations, etc., can solve the problem that viruses cannot be assembled

Active Publication Date: 2012-09-26
FRAUNHOFER USA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in these systems, when a polypeptide longer than 25 amino acids is fused to its capsid protein, the virus fails to assemble

Method used

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  • Virus like particles comprising target proteins fused to plant viral coat proteins
  • Virus like particles comprising target proteins fused to plant viral coat proteins
  • Virus like particles comprising target proteins fused to plant viral coat proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The construction of the heterologous vector expressing AIMV-CP fusion protein of embodiment 1

[0053] Target genes include different sex stages of malaria parasites, Plasmodium falciparum (Pfs25 (SEQ ID NO: 4), Pfs28 (SEQ ID NO: 5), and Pfs230 (SEQ ID NO: 49)), influenza virus Anwei strain blood Lectin (HA) globular domain (HA3A (SEQ ID NO: 6)), Influenza virus California strain HA globular domain (HA3C04 (SEQ ID NO: 7) and HA3C06 (SEQ ID NO: 8)), Influenza virus Indonesia strain HA The globular domain (HA3I (SEQ ID NO: 11)), the full-length HA of the Anhui strain (HAA or HAA1 (SEQ ID NO: 9)), and the full-length HA of the Indonesian strain (HAI or HAI1 (SEQ ID NO: 10)) are specific of cell surface proteins. Each target gene was cloned as an N-terminal fusion of an AIMV capsid protein (AIMV-CP, CP, or CPF), or an optimized AIMV capsid protein (CPO), using standard methods in molecular biology, wherein the optimized AIMV capsid The protein is encoded by a coding seque...

Embodiment 2

[0055] Example 2 Infiltration of Plants Using Expression Vectors Carrying Fusion Constructs

[0056] The expression vector produced in Example 1 was then introduced into Agrobacterium tumefaciens strain GV3101, and the resulting bacteria were grown overnight in minimal medium. The optical density of the culture was determined and the protein-expressing strain was mixed with the Agrobacterium strain expressing the silencing protein suppressor p19 in a 4:1 ratio to a final O.D. of 0.5. Agrobacterium solutions were introduced by manual infiltration into the aerial parts of 6-week-old, soil-grown N. benthamiana plants as previously described (Green et al., Biotechnol. J. 4:1-8, 2009).

[0057] Plant tissue samples were taken 3-7 days after infiltration to determine the expression level and solubility of the fusion protein. Samples were weighed and extracted in 3 volumes of extraction buffer (100 mM Na 2 HPO 4 , pH7.1; 2.5mM EDTA, pH8.0) to extract total soluble protein, extract...

Embodiment 3

[0058] Example 3 Isolation and purification of virus-like particles

[0059] On the day of maximum expression, leaves were harvested and homogenized in 3 volumes of phosphate extraction buffer with 0.5% TritonX-100 in a blender. The homogenate was stirred at 4°C for 30 minutes and then centrifuged at 5,000 xg for 30 minutes. The supernatant was filtered through miracloth and centrifuged at 15,000 xg for 1 to 1.5 hours. The supernatant was pelleted with PEG and centrifuged again at 15,000 x g for 30 min. Pellets were resuspended in phosphate buffer and frozen at -20°C. After thawing, the suspension was centrifuged at 30,000 xg for 30 minutes. Protein concentrations were determined by examining aliquots of the suspension by SDS-PAGE. The suspension was centrifuged at 60,000 xg for 2 hours in a Ti70 rotor. The pellet was resuspended in phosphate buffer. The resulting suspension was analyzed using SDS-PAGE and Coomassie blue staining. Protein concentration is determined col...

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PUM

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Abstract

Virus like particles comprising a fusion protein and substantially free of nucleic acid, wherein the fusion protein comprises a plant viral coat protein and a target protein, are provided. Immunogenic compositions comprising the virus like particles can be administered to subjects to induce protective immune responses in the subjects. Methods of producing the virus like particles are also provided.

Description

[0001] Cross References to Related Applications [0002] This patent application claims priority to US Provisional Application No. 61 / 243,774, filed September 18, 2009, and US Provisional Application No. 61 / 348,069, filed May 25, 2010, both of which are incorporated herein by reference in their entirety. field of invention [0003] The present invention relates generally to the field of recombinant vaccines. More specifically, the present invention relates to virus-like particles comprising a target protein fused to a plant viral capsid protein for use in vaccines, and methods of producing such virus-like particles. Background of the invention [0004] Plant viruses are effective tools for antigen production and delivery. Many important protein antigens have been expressed in transgenic plants, but the level of antigenic protein produced is relatively low. In an attempt to address this problem, plant viral capsid proteins have been designed to serve as carrier molecules fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61K39/015C12N7/04C07K14/11C07K14/445C12N15/82
CPCC12N2770/14023C07K2319/735C12N2710/10343A61K39/015C07K14/005C12N7/00C12N15/8258C07K14/445A61K39/145C12N2760/16122A61K2039/5258C12N2770/14022C12N2760/16234C12N2760/16134C12N2760/16222A61K39/12A61K2039/6075A61K2039/55505A61K2039/55577A61P31/16A61P33/02A61P37/04Y02A50/30
Inventor V·玉斯博夫C·E·法兰斯K·A·穆斯查克维迪姆·梅特维兰帝纳·梅特
Owner FRAUNHOFER USA
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