Computationally optimized broadly reactive antigens for H1N1 influenza

A technology of influenza and influenza virus, applied in the direction of virus antigen components, antisense single-stranded RNA virus, application, etc., can solve problems such as increasing the risk of infection

Inactive Publication Date: 2014-04-16
UNIVERSITY OF PITTSBURGH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Live virus vaccines should not be given to immunocompromised or pregnant patients because of the increased risk of infection in these patients

Method used

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  • Computationally optimized broadly reactive antigens for H1N1 influenza
  • Computationally optimized broadly reactive antigens for H1N1 influenza
  • Computationally optimized broadly reactive antigens for H1N1 influenza

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] Example 1: Generation of COBRA sequences for H1N1 influenza

[0157] Download the influenza A H1N1HA amino acid sequence from the NCBI Influenza Virus Resource Database. H1N1HA proteins from 1134 isolates from 1918-2011 were used to generate consensus sequences. Eleven different consensus sequences (SEQ ID NO: 1-11) were generated using the following method:

[0158] 1. COBRA Method 1 (1918-2005)

[0159] Use sources from 1918-1934 (8), 1935-1947 (13), 1948-1957 (12), 1977-1983 (68), 1984-1986 (9), 1987-1991 (12) , 1992-1999 (59) and 2000-2005 (263) isolates were sequenced according to the date of isolation (456), and then eight first consensus sequences were generated. By grouping the first consensus sequence by date, 4 second consensus sequences are generated, such as figure 1 shown. By aligning the four second consensus sequences, a final consensus sequence (third-level consensus sequence; SEQ ID NO: 1) was generated.

[0160] 2. COBRA Method 2 (1918-2005)

[...

Embodiment 2

[0181] Example 2: Preparation of influenza VLP and use of influenza VLP for immunization

[0182] The following methods can be used to generate and characterize influenza VLPs comprising optimized HA. Exemplary methods of immunizing mice, ferrets and macaques are also described below (see also, Giles and Ross, Vaccine 29(16):3043-3054, 2011).

[0183] vaccine preparation

[0184] 293T cells were transiently transfected with plasmids expressing M1, NA and optimized HA and incubated at 37°C for 72 hours. The sequences encoding M1, NA and HA can be codon optimized for expression in mammalian cells. The supernatant was collected, centrifuged at low speed to remove cell debris, and vacuum filtered through a 0.22 μm sterile filter. VLPs were purified by ultracentrifugation (100,000 x g through 20% glycerol, weight / volume) at 4°C for 4 hours. The pellet was then resuspended in PBS, pH 7.2, and stored in single-use aliquots at -80°C until use. Using Micro BCA TM Total protein co...

Embodiment 3

[0196] Example 3: Analysis of HA by COBRA H1N1 Method 1

[0197] This example describes the results that vaccination of mice with COBRA H1N1 influenza VLPs induced significant serum antibody titers to HAI and also shows that in vitro translation of COBRA H1N1 HA results in expression of the protein at the expected molecular weight.

[0198] HAI serum antibody titer

[0199] To assess whether influenza viruses containing COBRA Approach 1 H1N1 HA were able to elicit antibody responses in infected animals, VLPs comprising the COBRA Approach 1 HA (SEQ ID NO: 1 ) were generated as described in Example 2. The mice were inoculated with the VLP, and the serum was collected at the 3rd, 5th, 8th and 12th week after infection to determine the serum antibody titer of HAI. Antisera were tested against seasonal H1N1 influenza virus A / New Caledonia / 20 / 1999. Such as Figure 6 As shown, the HAI titer was detected at the 5th week after infection, and the HAI titer increased at the 8th and 12...

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Abstract

Described herein is the generation of optimized H1N1 influenza HA polypeptides for eliciting a broadly reactive immune response to H1N1 influenza virus isolates. The optimized HA polypeptides were developed through a series of HA protein alignments, and subsequent generation of consensus sequences, based on selected H1N1 viruses isolated from 1918-2011. Provided herein are optimized HlNl HA polypeptides, and compositions, fusion proteins and VLPs comprising the HA polypeptides. Further provided are codon-optimized nucleic acid sequences encoding the HA polypeptides. Methods of eliciting an immune response against influenza virus in a subject are also provided by the present disclosure.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application No. 61 / 498,800, filed June 20, 2011, which is incorporated herein by reference in its entirety. technical field [0003] The present disclosure relates to optimized influenza hemagglutinin proteins capable of eliciting broadly reactive immune responses against H1N1 virus isolates and their use as vaccines. Background technique [0004] Influenza viruses are members of the Orthomyxoviridae family. There are three subtypes of influenza virus, named influenza A, influenza B, and influenza C. The influenza virion contains a segmented negative-sense RNA genome encoding the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (M1), proton ion channel protein (M2), nuclear protein (NP), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA) and nonstructural protein 2 (NS2). HA, NA, M1 and M2 are membran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/33C12N15/44C12N15/63
CPCC12N2760/16122A61K39/145A61K39/12A61K2039/521A61K2039/55505C12N2760/16123A61K2039/5258C12N15/63C12N2760/16134C07K14/005A61P31/16A61P37/04C12N7/00
Inventor T·M·罗斯B·M·吉尔斯C·J·克里瓦尔
Owner UNIVERSITY OF PITTSBURGH
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