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Modified influenza hemagglutinin proteins and uses thereof

A kind of hemagglutinin, protein technology, be used in the immunogenic composition such as vaccine field of treating and/or preventing influenza infection

Inactive Publication Date: 2013-11-06
NOVAVAX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

VLPs lack viral DNA or RNA genomes, but retain the three-dimensional structure of real viruses

Method used

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  • Modified influenza hemagglutinin proteins and uses thereof
  • Modified influenza hemagglutinin proteins and uses thereof
  • Modified influenza hemagglutinin proteins and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0153] Example 1: Generation of influenza virus-like particles (VLP) with modified hemagglutinin (HA) protein

[0154] This example describes the generation of influenza H5N1 VLPs prepared with HA protein modified to eliminate one or more glycosylation sites. To construct a modified HA protein, one or both of the NX(T / S) sequences at amino acid positions 170-172 and 181-183 of the A / Indonesia / 05 / 2005 (H5N1) HA1 (SEQ ID NO: 2) protein were mutated. By( figure 1 ). Specifically, threonine was mutated to alanine at positions 172 and / or 183 of the HA1 protein.

[0155] After modifying the HA gene, the modified HA gene was co-expressed with the NA and M1 genes derived from A / Indonesia / 5 / 2005 (H5N1). The HA, NA, and M1 genes were expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression system. Expression products of infected Sf9 cells were characterized by SDS-PAGE analysis. Such as figure 2 As shown, HA, NA, and M1 proteins were detected with ex...

Embodiment 2

[0156] Example 2: Influenza VLPs with Modified Hemagglutinin (HA) Protein Retain Hemagglutinin and Neuraminidase active

[0157] In this example, the hemagglutinin and neuraminidase activities of the HA and NA proteins in wild-type A / Indonesia / 05 / 2005 (H5N1) VLPs were compared with (SEQ ID NO: 2) Those same activities of the H5N1 VLP of the HA protein with one or more glycosylation sites at positions 170-172 and 181-183 were compared.

[0158] To determine the hemagglutination activity of influenza VLPs, two-fold serial dilutions of sucrose gradient fractions containing wild-type influenza VLPs or influenza VLPs comprising modified HA protein were prepared. Next, these VLPs were mixed with guinea pig erythrocytes in PBS and incubated at 4°C for 1-16 hours. The degree of hemagglutination was assessed visually and the highest dilution capable of agglutinating guinea pig erythrocytes was determined. The highest hemagglutination titer observed for wild type influenza VLP was...

Embodiment 3

[0160] Example 3: Using NetNGlyc 1.0 software to identify N-linked glycosylation sites

[0161] In this example, NetNGlyc predictive analysis software was used to identify N-linked glycosylation sites in the top of the globular head in three seasonal influenza strains.

[0162] The first strain analyzed in this example was A / California / 07 / 2009 (H1N1). The NetNGlyc prediction software identified glycosylation sites at positions 28 (accompanied by the amino acid sequence "NSTD"), 40 (NVTV), 304 (NTSL), and 557 (NGSL). Of these sites, none were located in the "vertical" region. Thus, HA from pandemic A / California / 07 / 2009 (H1N1) did not contain any glycosylation sites in the region located near the top of the HA globular head.

[0163] The second strain analyzed in this example was A / Perth / 16 / 2009 (H3N2). NetNGlyc prediction software identified glycans at positions 24 (NSTA), 38 (NGTI), 79 (NCTL), 142 (NWTG), 149 (NGTS), 181 (NVTM), 262 (NSTG), and 301 (NGSI) chemical site...

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Abstract

The present invention is generally directed to modified influenza hemagglutinin (HA) proteins and methods for making and using them, including their use in immunogenic compositions such as vaccines for the treatment and / or prevention of influenza infection.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application Serial No. 61 / 426,390, filed December 22, 2010, which is hereby incorporated by reference in its entirety for all purposes. technical field [0003] In general, the present invention relates to modified influenza hemagglutinin (HA) proteins and methods for producing and using them, including their use in immunogenic compositions such as vaccines for the treatment and / or prevention of influenza infection . [0004] Description of electronically submitted text files [0005] The contents of the text file submitted electronically with this document are incorporated in its entirety by reference: Computer-readable copy of the sequence listing (file name: NOVV_046_00US_SeqList_ST25.txt, date of record: October 29, 2010, file size: 10 kilobytes ). Background of the invention [0006] Influenza virus is a member of the Orthomyxoviridae family and contains a segm...

Claims

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Application Information

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IPC IPC(8): A61K39/145C12N15/44
CPCC12N2760/16122C12N7/00A61K2039/5258C12N2799/026C12N2760/16134C12N2760/16123A61K39/00A61P31/16A61P37/04C12N15/11A61K39/145
Inventor G.史密斯Y.刘M.马萨里R.辛维
Owner NOVAVAX
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