Influenza virus-like particles (VLPS) comprising hemagglutinin produced within a plant

a technology of viruslike particles and hemagglutinin, which is applied in the direction of immunological disorders, drug compositions, peptides, etc., can solve the problems of increasing the risk of emergence or the transmission to humans of a subtype endemic in animals, and the risk of a new subtype or a new subtype is always present, so as to facilitate the capture of glycoprotein antigens, strengthen the immune response, and enhance the immune respons

Inactive Publication Date: 2010-09-23
MEDICAGO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0045]The production of VLPs in plants presents several advantages over the production of these particles in insect cell culture. Plant lipids can stimulate specific immune cells and enhance the immune response induced. Plant membranes are made of lipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and also contain glycosphingolipids that are unique to plants and some bacteria and protozoa. Sphingolipids are unusual in that they are not esters of glycerol like PC or PE but rather consist of a long chain amino alcohol that forms an amide linkage to a fatty acid chain containing more than 18 carbons. PC and PE as well as glycosphingolipids can bind to CD1 molecules expressed by mammalian immune cells such as antigen-presenting cells (APCs) like dendritic cells and macrophages and other cells including B and T lymphocytes in the thymus and liver (Tsuji M., 2006). Furthermore, in addition to the potential adjuvant effect of the presence of plant lipids,...

Problems solved by technology

Influenza pandemics are usually caused by highly transmittable and virulent influenza viruses, and can lead to elevated levels of illness and death globally.
Presently, the risk of the emergence of a new subtype, or of the transmission to humans of a subtype endemic in animals, is always present.
In many cases, this bird flu can result in mortality rates approaching 100% within 48 hours.
Generally, the number of vaccine doses produced each year is not sufficient to vaccinate the world's population.
It is evident that current worldwide production of influenza vaccine would be insufficient in the face of a worldwide flu pandemic.
Even if the necessary annual production could somehow be met in a given year, the dominant strains change from year to year, thus stockpiling at low-need times in the year is not practical.
While these methods may produce useful vaccines, they do not provide a solution to the need for high-volume, low cost and fast production of vaccines in the scale necessary to meet the global need in a normal year, and would almost certainly be insufficient in the face of a pandemic.
For highly pathogenic influenza strains, the production of whole virus vaccines either requires confinement procedures or the resulting vaccines do not exactly match the genetic sequence of the circulating virus.
In the case of live-attenuated vaccines, there is still a risk that the administered vaccine can recombine with an influenza virus from the host, leading to a new inf...

Method used

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  • Influenza virus-like particles (VLPS) comprising hemagglutinin produced within a plant
  • Influenza virus-like particles (VLPS) comprising hemagglutinin produced within a plant
  • Influenza virus-like particles (VLPS) comprising hemagglutinin produced within a plant

Examples

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example 1

Transient Expression of Influenza Virus A / Indonesia / 5 / 05 (H5N1) Hemagglutinin by Agroinfiltration in N. benthamiana Plants

[0292]The ability of the transient expression system to produce influenza hemagglutinin was determined through the expression of the H5 subtype from strain A / Indonesia / 5 / 05 (H5N1). As presented in FIG. 11, the hemagglutinin gene coding sequence (Acc. #EF541394), with its native signal peptide and transmembrane domain, was first assembled in the plastocyanin expression cassette-promoter, 5′UTR, 3′UTR and transcription termination sequences from the alfalfa plastocyanin gene—and the assembled cassette (660) was inserted into to a pCAMBIA binary plasmid. This plasmid was then transfected into Agrobacterium (AGL1), creating the recombinant strain AGL1 / 660, which was used for transient expression.

[0293]N. benthamiana plants were infiltrated with AGL1 / 660, and the leaves were harvested after a six-day incubation period. To determine whether H5 accumulated in the agroin...

example 2

Characterization of Hemagglutinin-Containing Structures in Plant Extracts Using Size Exclusion Chromatography

[0295]The assembly of plant-produced influenza hemagglutinin into high molecular weight structures was assessed by gel filtration. Crude protein extracts from AGL1 / 660-infiltrated plants (1.5 mL) were fractionated by size exclusion chromatography (SEC) on Sephacryl™ S-500 HR columns (GE Healthcare Bio-Science Corp., Piscataway, N.J., USA). Elution fractions were assayed for their total protein content and for HA abundance using immunodetection with anti-HA antibodies (FIG. 13A). As shown in FIG. 13A, Blue Dextran (2 MDa) elution peaked early in fraction 10 while the bulk of host proteins was retained in the column and eluted between fractions 14 and 22. When proteins from 200 μL of each SEC elution fraction were concentrated (5-fold) by acetone-precipitation and analyzed by Western blotting (FIG. 15A, H5), hemagglutinin (H5) was primarily found in fractions 9 to 14 (FIG. 13B)...

example 3

Isolation of H5 Structures by Centrifugation in Sucrose Gradient and Observation Under Electron Microscopy

[0299]The observation of hemagglutinin structure under electron microscopy (EM) required a higher concentration and purity level than that obtained from SEC on crude leaf protein extracts. To allow EM observation of H5 structures, a crude leaf protein extract was first concentrated by PEG precipitation (20% PEG) followed by resuspension in 1 / 10 volumes of extraction buffer. The concentrated protein extract was fractionated by S-500 HR gel filtration and elution fractions 9, 10, and 11 (corresponding to the void volume of the column) were pooled and further isolated from host proteins by ultracentrifugation on a 20-60% sucrose density gradient. The sucrose gradient was fractionated starting from the top and the fractions were dialysed and concentrated on a 100 NMWL centrifugal filter unit prior to analysis. As shown on the Western blots and hemagglutination results (FIG. 14A), H5...

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Abstract

A method for synthesizing influenza virus-like particles (VLPs) within a plant or a portion of a plant is provided. The method involves expression of influenza HA in plants and the purification by size exclusion chromatography. The invention is also directed towards a VLP comprising influenza HA protein and plants lipids. The invention is also directed to a nucleic acid encoding influenza HA as well as vectors. The VLPs may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.

Description

FIELD OF INVENTION[0001]The present invention relates to the production of virus-like particles. More specifically, the present invention is directed to the production of virus-like particles comprising influenza antigens.BACKGROUND OF THE INVENTION[0002]Influenza is the leading cause of death in humans due to a respiratory virus. Common symptoms include fever, sore throat, shortness of breath, and muscle soreness, among others. During flu season, influenza viruses infect 10-20% of the population worldwide, leading to 250-500,000 deaths annually[0003]Influenza viruses are enveloped virus that bud from the plasma membrane of infected mammalian cells. They are classified into types A, B, or C, based on the nucleoproteins and matrix protein antigens present. Influenza type A viruses may be further divided into subtypes according to the combination of hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins presented. HA governs the ability of the virus to bind to and penetrate t...

Claims

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Application Information

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IPC IPC(8): A61K39/145C07H21/04C07K14/11A61P31/16C12N7/00
CPCA61K39/145A61K2039/5258A61K2039/543C07K14/005C12N7/00C12N15/8257A61K2039/58C12N2760/16122C12N2760/16123C12N2760/16134A61K2039/517A61K2039/55505A61K2039/55583C12N15/8258A61K39/12A61P31/00A61P31/16A61P37/00A61P37/04C07K14/11C12N15/82A61K2039/55588C07K2319/02C07K2319/03C12N9/90C12N2760/16022C12N2760/16133C12N2760/16151C12N2760/16171C12N2760/16222C12N2760/16223C12N2760/16234C12Y503/04001G06F9/505G06F2209/508
Inventor D'AOUST, MARC-ANDRECOUTURE, MANONORS, FREDERICTREPANIER, SONIALAVOIE, PIERRE-OLIVIERDARGIS, MICHELEVEZINA, LOUIS-PHILIPPELANDRY, NATHALIE
Owner MEDICAGO INC
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